U.S. patent application number 11/543882 was filed with the patent office on 2007-08-23 for extranuclear rna splicing in neuronal dendrites.
This patent application is currently assigned to The Trustees of the University of Pennsylvania. Invention is credited to James Eberwine, Jason Glanzer, Kevin Miyashiro.
Application Number | 20070196921 11/543882 |
Document ID | / |
Family ID | 35150003 |
Filed Date | 2007-08-23 |
United States Patent
Application |
20070196921 |
Kind Code |
A1 |
Eberwine; James ; et
al. |
August 23, 2007 |
Extranuclear RNA splicing in neuronal dendrites
Abstract
The present invention relates to methods of synaptic network
remodeling by means of extranuclear RNA splicing. The present
invention also provides methods of extranuclear RNA splicing, and
methods of protein translation based on extranuclear RNA
splicing.
Inventors: |
Eberwine; James;
(Philadelphia, PA) ; Miyashiro; Kevin;
(Philadelphia, PA) ; Glanzer; Jason; (Omaha,
NE) |
Correspondence
Address: |
DRINKER BIDDLE & REATH;ATTN: INTELLECTUAL PROPERTY GROUP
ONE LOGAN SQUARE
18TH AND CHERRY STREETS
PHILADELPHIA
PA
19103-6996
US
|
Assignee: |
The Trustees of the University of
Pennsylvania
|
Family ID: |
35150003 |
Appl. No.: |
11/543882 |
Filed: |
October 5, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US05/11637 |
Apr 7, 2005 |
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|
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11543882 |
Oct 5, 2006 |
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60560039 |
Apr 7, 2004 |
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Current U.S.
Class: |
435/455 ;
435/368; 435/375 |
Current CPC
Class: |
C12N 2510/00 20130101;
C12N 15/10 20130101; C12N 15/1079 20130101; A61K 48/0008 20130101;
C12N 5/0619 20130101 |
Class at
Publication: |
435/455 ;
435/368; 435/375 |
International
Class: |
C12N 15/09 20060101
C12N015/09; C12N 5/08 20060101 C12N005/08 |
Claims
1. A method of remodeling a dendrite, said method comprising the
steps of: a. transfecting a dendrite with an RNA comprising at
least one intron, wherein said dendrite comprises at least one
component of a spliceosome and further wherein said component of a
spliceosome is capable of splicing an RNA; b. allowing said RNA
comprising at least one intron to be spliced by said spliceosome
components; and c. allowing said spliced RNA to be translated in
said dendrite; wherein said dendrite is thereby remodeled as a
consequence of said translation.
2. A method of remodeling a dendrite interaction, said method
comprising the steps of: a. transfecting a dendrite with an RNA
comprising at least one intron, wherein said dendrite comprises at
least one component of a spliceosome and further wherein said
component of a spliceosome is capable of splicing an RNA; b.
allowing said RNA comprising at least one intron to be spliced by
said spliceosome components; and c. allowing said spliced RNA to be
translated in said dendrite; wherein said dendrite interaction is
thereby remodeled as a consequence of said translation.
3. A method of remodeling a synaptic network comprising interaction
with at least one dendrite, said method comprising the steps of: a.
transfecting a dendrite with an RNA comprising at least one intron,
wherein said dendrite comprises at least one component of a
spliceosome and further wherein said component of a spliceosome is
capable of splicing an RNA; b. allowing said RNA comprising at
least one intron to be spliced by said spliceosome components; and
c. allowing said spliced RNA to be translated in said dendrite;
wherein said synaptic network is thereby remodeled as a consequence
of said translation.
4. A method of splicing an RNA, said method comprising the steps
of: a. providing an isolated dendrite comprising at least one
component of a spliceosome, wherein said component is capable of
splicing an RNA; and b. transfecting said dendrite with an RNA
comprising at least one intron; wherein said RNA comprising at
least one intron is spliced by said spliceosome components.
5. The method of claim 1, wherein said dendrite is a component of a
neuron.
6. The method of claim 1, wherein said dendrite is an isolated
dendrite.
7. A method of splicing an RNA, said method comprising the steps
of: a. providing an isolated cell comprising at least one component
of a spliceosome in the cytoplasm, wherein said component is
capable of splicing an RNA; and b. transfecting said cell with an
RNA comprising at least one intron; wherein said RNA comprising at
least one intron is spliced by said spliceosome components in said
cytoplasm.
8. The method of claim 1, wherein RNA splicing donor/acceptor pairs
are selected from the group consisting of canonical, atypical and
cryptic.
9. The method of claim 1, wherein said RNA comprising at least one
intron is a pre-RNA.
10. The method of claim 9, wherein said pre-RNA is a pre-mRNA.
11. The method of claim 1, wherein said RNA comprising at least one
intron is derived from a nucleic acid comprising a construct
selected from the group consisting of pEGFP-N1, pDsRed-N1,
SF1/mBBP-GFP, U2AF65-GFP, a GFP construct, a DsRed construct, a
histone 2B-YFP construct.
12. The method of claim 1, wherein at least one spliceosome
component is selected from the group consisting of Y14, Magoh, RNPS
1, SC-35, SF2, U2AF65, Smith antigen, pan-SR antigen, U1 snRNP, U2
snRNP, U4 snRNP, U5 snRNP, U6 snRNP.
13. A method of translating an RNA, said method comprising the
steps of: a. providing an isolated dendrite comprising at least one
component of a spliceosome, wherein said component is capable of
splicing an RNA; and b. transfecting said dendrite with an RNA
comprising at least one intron, wherein said RNA is spliced by said
spliceosome components; further wherein said spliced RNA is
translated.
14. A method of translating an RNA, said method comprising the
steps of: a. providing an isolated cell comprising at least one
component of a spliceosome in the cytoplasm, wherein said component
is capable of splicing an RNA; and b. transfecting said cell with
an RNA comprising at least one intron, wherein said RNA is spliced
by said spliceosome components in said cytoplasm; further wherein
said spliced RNA is translated.
15. A method of splicing an RNA, said method comprising the steps
of: a. providing an isolated synaptoneurosome comprising at least
one component of a spliceosome, wherein said component is capable
of splicing an RNA; and b. contacting said synaptoneurosome with an
RNA comprising at least one intron; wherein said RNA comprising at
least one intron is spliced by said spliceosome components.
16. A method of translating an RNA, said method comprising the
steps of: a. providing an isolated synaptoneurosome comprising at
least one component of a spliceosome, wherein said component is
capable of splicing an RNA; b. contacting said synaptoneurosome
with an RNA comprising at least one intron, wherein said RNA is
spliced by said spliceosome components; and c. contacting said
spliced RNA with a composition capable of translating an RNA under
conditions suitable for translating an RNA.
Description
BACKGROUND OF THE INVENTION
[0001] In a living cell, such as a neuron, from the moment a
primary RNA transcript is complete to the actual expression of the
protein encoded by the transcript, multiple cellular events and
mechanisms occur, including pre-RNA splicing, RNA editing,
shuttling of the mRNA between the nucleus and the cytoplasm, and
processes that ensure the stability and translational control of
the trafficked mRNAs. Each of these events provides opportunities
for the cell to regulate gene expression at the RNA level.
[0002] Each neuron is comprised of a nucleus within a body, or
soma, a long fiber called the axon, and a varying number of
branching fibers called dendrites, which extend out to other
neurons. A single neuron can make numerous contacts with other
neurons and tissues. For example, every new thought process is
handled by a new set of synaptic connections. Memory itself is a
set of synaptic connections engraved in the network of neurons.
[0003] Dendrites are specialized extensions of the neuronal soma
that contain components of the cellular machinery involved in RNA
and protein metabolism. A subset of mRNAs are trafficked to
dendrites through their association with RNA binding proteins
(RBPs). Some of these RBPs function in the nucleus as mediators of
pre-RNA splicing.
[0004] The functional properties of neurites, including dendrites
and axons, have been extensively examined since the discovery of
protein synthetic machinery in dendrites, including ribosomes and
membranous constituents of the endoplasmic reticulum and golgi
apparatus (Bodian, 1965, Proc. Natl. Acad. Sci. U.S.A., 53:418-425;
Steward et al., 1983, Res. 58:131-6; Torre et al., 1996, J.
Neurosci. 16:5967-78; Gardiol et al., 1999, J. Neurosci.
19:168-79). Increasingly, more detailed molecular analyses of
dendrites have shown that a subset of cellular RNAs are transported
into dendrites where they can be translated into protein at
specialized areas following synaptic stimulation (Aakalu et al.,
2001, Neuron 30:489-502; Bassell et al., 1998, J. Neurosci.
18:251-65; Crino et al., 1996, Neuron 17:1173-87; Huber et al.,
2000, Science 288:1254-7; Job et al., 2001, Proc. Natl. Acad. Sci.
U.S.A 98:13037-42; Martin et al., 1997, Cell, 91:927-38). In the
cytoplasm, the intracellular transport, stability, and translation
of RNA are regulated by RNA binding proteins (RBPs) (Spirin et al.,
1979, Mol. Biol. Rep. 5:53-57). RBP-RNA interactions typically
occur through conserved motifs in RBPs that associate with cis
acting sequences or secondary structures in RNA.
[0005] Recently, other RBPs thought to function only in the nucleus
have also been localized in the cytoplasm. These include RNA
editing enzymes (e.g. double stranded RNA adenosine deaminase)
(Strehblow et al., 2002, Mol. Biol. Cell, 13:3822-35) as well as
some of the highly conserved constituents of the spiceosome (e.g.
the survival of motor neuron proton (Fan et al., 2002, Hum. Mol.
Genet. 11: 1605-14) and a variety of heterogeneous nuclear
ribonucleoproteins (hnRNPs) (Pinol-Roma, 1997, Semin. Cell Dev.
Biol. 8:57-63). Some auxiliary components of the spliceosome, such
as the splicing factor SAM68, are present within the
somatodendritric compartment of neurons as well (Staley et al.,
1998, Cell 92:315-26; Jurica et al., 2003, Mol. Cell 12:5-14;
Grange et al., 2004, J. Neurosci. Res., 75:654-66). The presence of
these proteins in a non-nuclear compartment suggests that they
either serve a unique functional role outside of the nucleus or
their known functional activity can occur within this subcellular
compartment.
[0006] The spliceosome, which catalyses the ATP-dependent removal
of introns from nuclear pre-RNA, is a multi-megadalton complex of
proteins and small nuclear RNAs (snRNA) (Staley et al., 1998, Cell
92:315-26; Jurica et al., 2003, Mol. Cell 12:5-14). Even in the
nucleus, the distribution of pre-RNA splicing factors is not
uniform. Rather, within discrete sites of concentration and lower
levels of factors diffusely dispersed throughout the nucleoplasm,
speckles (splicing factor compartments) can be readily identified
with an antibody against the spliceosome assembly factor SC-35
(Lamond et al., 2003, Nat. Rev. Mol. Cell Biol. 4:605-12).
[0007] Despite the existing knowledge of these nuclear factors in
the cytoplasm, the current state of the art does not definitively
attribute function or role to the presence of RBPs and spliceosome
components in the cytoplasm. A greater understanding of the
regulation, metabolism and growth of cells will enable more
accurate and more useful control and manipulation of cells. The
development of such tools can enable more precise, targeted
therapies and treatments of all mammals, and in particular, of
humans. Therefore, there exists a need for a better understanding
of the function and role of RBPs and spliceosome components in the
cytoplasm in order to facilitate the controlled manipulation of
cells. The present invention addresses and meets these needs.
SUMMARY OF THE INVENTION
[0008] The present invention features a method of remodeling a
dendrite comprising the steps of transfecting a dendrite with an
RNA comprising at least one intron, wherein the dendrite comprises
at least one component of a spliceosome and further wherein the
component of a spliceosome is capable of splicing an RNA. The
method also includes the step of allowing the RNA comprising at
least one intron to be spliced by the spliceosome components and
allowing the spliced RNA to be translated in the dendrite, wherein
the dendrite is thereby remodeled as a consequence of the
translation.
[0009] The invention also features a method of remodeling a
dendrite interaction comprising the steps of transfecting a
dendrite with an RNA comprising at least one intron, wherein the
dendrite comprises at least one component of a spliceosome and
further wherein the component of a spliceosome is capable of
splicing an RNA. The method also includes the step of allowing the
RNA comprising at least one intron to be spliced by the spliceosome
components and allowing the spliced RNA to be translated in the
dendrite, wherein the dendrite interaction is thereby remodeled as
a consequence of the translation.
[0010] The present invention further features a method of
remodeling a synaptic network comprising interaction with at least
one dendrite comprising the steps of transfecting a dendrite with
an RNA comprising at least one intron, wherein the dendrite
comprises at least one component of a spliceosome and further
wherein the component of a spliceosome is capable of splicing an
RNA. The method also includes the step of allowing the RNA
comprising at least one intron to be spliced by the spliceosome
components and allowing the spliced RNA to be translated in the
dendrite, wherein the synaptic network is thereby remodeled as a
consequence of the translation.
[0011] In one aspect of the invention, a dendrite is a component of
a neuron. In another aspect, a dendrite is an isolated
dendrite.
[0012] The invention also provides a method of splicing an RNA
comprising the steps of providing an isolated dendrite comprising
at least one component of a spliceosome, wherein the component is
capable of splicing an RNA, and transfecting the dendrite with an
RNA comprising at least one intron, wherein the RNA comprising at
least one intron is spliced by the spliceosome components.
[0013] The invention further provides a method of splicing an RNA
comprising the steps of providing an isolated cell comprising at
least one component of a spliceosome in the cytoplasm, wherein the
component is capable of splicing an RNA, and transfecting a cell
with an RNA comprising at least one intron, wherein the RNA
comprising at least one intron is spliced by the spliceosome
components in the cytoplasm.
[0014] In one aspect of the invention, the RNA splicing
donor/acceptor pairs are selected from the group consisting of
canonical, atypical and cryptic. In another aspect of the
invention, an RNA comprising at least one intron is a pre-RNA. In
another aspect, a pre-RNA is a pre-mRNA.
[0015] In one embodiment of the invention, an RNA comprising at
least one intron is derived from a nucleic acid comprising a
construct selected from the group consisting of pEGFP-N1,
pDsRed-N1, SF1/mBBP-GFP, U2AF65-GFP, a GFP construct, a DsRed
construct, a histone 2B-YFP construct.
[0016] In another embodiment of the invention, at least one
spliceosome component is selected from the group consisting of Y14,
Magoh, RNPS1, SC-35, SF2, U2AF65, Smith antigen, pan-SR antigen, U1
snRNP, U2 snRNP, U4 snRNP, U5 snRNP, U6 snRNP.
[0017] The invention also features a method of translating an RNA,
comprising the steps of providing an isolated dendrite comprising
at least one component of a spliceosome, wherein the component is
capable of splicing an RNA, and transfecting the dendrite with an
RNA comprising at least one intron, wherein the RNA is spliced by
the spliceosome components.
[0018] The invention features a method of translating an RNA,
comprising the steps of providing an isolated cell comprising at
least one component of a spliceosome in the cytoplasm, wherein the
component is capable of splicing an RNA, and transfecting the cell
with an RNA comprising at least one intron, wherein the RNA is
spliced by the spliceosome components in the cytoplasm. The method
further includes translation of the spliced RNA.
[0019] The present invention also provides a method of splicing an
RNA, comprising the steps of providing an isolated synaptoneurosome
comprising at least one component of a spliceosome, wherein the
component is capable of splicing an RNA, contacting said
synaptoneurosome with an RNA comprising at least one intron,
wherein the RNA comprising at least one intron is spliced by the
spliceosome components.
[0020] The invention also features a method of translating an RNA,
comprising the steps of providing an isolated synaptoneurosome
comprising at least one component of a spliceosome, wherein the
component is capable of splicing an RNA, contacting the
synaptoneurosome with an RNA comprising at least one intron,
wherein the RNA is spliced by the spliceosome components, and
contacting the spliced RNA with a composition capable of
translating an RNA under conditions suitable for translating an
RNA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] For the purpose of illustrating the invention, there are
depicted in the drawings certain embodiments of the invention.
However, the invention is not limited to the precise arrangements
and instrumentalities of the embodiments depicted in the
drawings.
[0022] FIG. 1 is a chart depicting sequences of
dendritically-spliced and synaptoneurosome-spliced RNAs.
[0023] FIG. 2, comprising FIGS. 2A through 2L, is a series of
images demonstrating coexistence of pre-spliceosome proteins in
neuronal dendrites. FIGS. 2A through 2C are images of cultured
hippocampal neurons labeled with Qdots 525/605 for detection of
SC-35, U2AF and merged of FIGS. 2A and 2B, respectively. FIGS. 2D
through 2F are images of cultured hippocampal neurons labeled with
Qdots 525/605 for detection of SC-35, Sm antigen and merged of
FIGS. 2D and 2E, respectively. FIGS. 2G through 2I are images of
cultured hippocampal neurons labeled with Qdots 525/605 for
detection of SC-35, SF2, and merged of FIGS. 2G and 2H,
respectively. FIGS. 2J through 2L are images of cultured
hippocampal neurons labeled with Qdots 525/605 for detection of SR,
SF2, and merged of FIGS. 2J and 2K, respectively. MAP2 protein was
detected in all samples via Cy5 immuno-detection and is shown in
the inset panel in FIG. 2A. Qdot secondary antibodies alone without
primary antibodies (see inset in FIGS. 2G and 2H) do not produce an
observable signal in these cells.
[0024] FIG. 3, comprising FIGS. 3A through 3F, is a series of
images demonstrating detection of U1 snRNA in dendrites. Neurons
were subjected to in situ hybridization (ISH) with antisense
digoxigenin-labeled U1 RNA (FIG. 3A) and a competition control
where an excess of unlabelled U1 antisense RNA was hybridized to
the section followed by washing and addition of digoxigenin-labeled
RNA (FIG. 3B). Control ISH used digoxygeninlabeled GAD65 antisense
RNA (FIG. 3E). Hybridized RNA was detected by alkaline phosphatase
conjugated anti-digoxigenin antibody in the presence of NBT/BCIP. A
strong nuclear presence of U1 RNA along with moderate staining
within several dendrites was observed. Without wishing to be bound
by any particular theory, background nuclear staining in FIG. 3B is
attributed to hybridization to U1 genes and pseudogenes. FIGS. 3C
and 3D show MAP2 immunofluorescence of antisense and competition
control U1 in situ hybridizations, respectively, in order to
identify the dendrites on these neurons. FIG. 3E depicts that GAD65
RNA is predominantly localized to the cell soma. There was little
to no observable GAD65 ISH signal in the neuronal processes (as
observed by phase contrast in FIG. 3F). The bright halo surrounding
the neuron in phase-contrast (FIG. 3F) is a characteristic of
healthy neurons whether live or fixed.
[0025] FIG. 4, comprising FIGS. 4A through 4O, is a series of
images demonstrating distribution of selected components of
spliceosomal subcomplexes A, B*, and C. Olympus Fluoview FV1000
images of primary hippocampal cells show the subcellular
localization of the selected components of each spliceosome
subcomplex. For subcomplex A, comprising FIGS. 4A through 4D,
SF1/mBBP-GFP (FIG. 4A) and U2AF65-GFP (FIG. 4C) were used. Lower
magnification whole cell views were stained for MAP2 with a Cy3
secondary antibody (FIGS. 4A-1 and 4C-1). A hatched box denotes the
area highlighted by higher magnification views. Higher
magnification of SFI/mBBP-GFP (FIGS. 4B and 4B-1) and U2AF65-GFP
(FIGS. 4D and 4D-1) and their corresponding MAP2 immunofluorescence
illustrate the presence of small puncta expressed throughout the
dendrite. For subcomplex B*, immunofluorescence of anti-PSF
monoclonal antibody in whole cell (FIG. 4E-1) and higher
magnification view (FIG. 4F-1) are shown. Corresponding MAP2
immunofluorescence is shown using Alexa 488 secondary antibody for
each of these views (FIGS. 4E and 4F). For subcomplex C,
immunofluorescence of anti-Aly/REF monoclonal antibody with Cy3
secondary antibody in low (FIG. 4G-1) and high (FIG. 4H-1)
magnification views is shown. Corresponding MAP2 immunofluorescence
is shown using Alexa 488 (FIGS. 4G and 4H). GFP fusion constructs
for Magoh, UAP56, or Y14 were used. Whole cell (FIGS. 4I, 4L, and
4N) or higher magnification of dendritic fields (FIGS. 4, 4J, 4M,
and 4O) show the diffusely and more concentrated puncta within the
dendroplasm. MAP2 immunofluorescence was visible in the red channel
in both low (FIGS. 41-1, 4L-1, and 4N-1) and high magnification
(FIGS. 4M-1 and 4O-1). FIG. 4K depicts a whole cell view of a glial
cell tranfected with the Magoh-GFP construct. It was observed that
Magoh-GFP expression and U2AF65-GFP show low levels of GFP
expression with a readily visible nuclear distribution and diffused
GFP fluorescence throughout the glial cytoplasm.
[0026] FIG. 5, comprising FIGS. 5A and 5B, demonstrates detection
of CDC pre-RNA splicing in an Sm-antigen positive dendrite. FIG. 5A
depicts an inset of an area harvested for detection of spliced
products. The yellow appearance of the neuronal nuclei is
attributed to blending of the green nuclear fluorescent
immunostaining of Sm protein and the red cytoplasmic fluorescent
immunostaining of the MAP2 protein. FIG. 5B is a schematic of a
methodology for CDC pre-RNA splicing. Briefly, after RNA
transfection, in situ transcription (IST) of CDC RNA and
immunofluorescence (IMF) detection of Sm granules was performed. Sm
proteins positive dendrites were harvested and their nucleic acid
components were amplified for spliced CDC RNA content. These PCR
amplicons were cloned and sequenced. An intron/exon sequence was
derived from the dendrite harvested in FIG. 5A using this
methodology.
[0027] FIG. 6 is a image depicting a western blot detection of
splicing proteins in synaptoneurosomes. Homogenates of whole brain
(WB) and synaptoneurosomes (SN) were probed with the corresponding
antibody. Numbers on the left depict the relative molecular weight
of the protein bands (KDa). The tubulin signal is included at the
bottom of the autoradiograms to demonstrate that the same amount of
protein was loaded for the whole brain and synaptoneurosome
fractions.
[0028] FIG. 7, comprising FIGS. 7A through 7B, demonstrates protein
translation from FLAG-tagged CDC pre-mRNA substrate in isolated
dendrites. FIGS. 7A, 7D, and 7G depict phase contrast images of
whole neurons before cell body removal. Corresponding phase
contrast images of isolated dendrites transfected with; no RNA
(FIG. 7B), mature CDC RNA (FIG. 7E), or unspliced, CDC pre-RNA
(FIG. 7H), and subsequent immunodetection of protein translation
with antibody for FLAG and DAB visualization are shown in FIGS. 7C,
7F, and 7I, respectively. Black arrows indicate isolated,
transfected dendrites before and after immunodetection of FLAG
protein translation. White arrows point out areas previously
occupied by cell bodies before dissection.
[0029] FIG. 8, comprising FIGS. 8A through 8J, is a series of
images demonstrating that FLAG protein can be translated from
alternatively spliced CDC RNAs. FIGS. 8G, 8H, 8I, and 8J depict
schematics of CDC pre-RNA and processed CDC RNAs used in the
isolated dendrite transfection assay. CDC RNAs were synthesized
from alternatively spliced variants 1, 6, and 45 (see FIG. 1, also
set forth in Table 1) and these RNAs were transfected into isolated
dendrites and subjected to immunohistochemistry. FLAG
immunoreactivity was visualized with DAB. Red, represents premature
termination codon produced in first reading frame by alternative
splicing. White, represents the initiator methionine of the second
cistron. FIGS. 8A, 8C, and 8E are brightfield images of isolated
dendrites transfected with 236 bp, 181 bp, and 146 bp RNAs,
respectively and subsequent phase contrast images are depicted
FIGS. 8B, 8D, and 8F, respectively. Black arrows indicate isolated
dendrites and areas of varied FLAG immunoreactivity.
[0030] FIG. 9 is a schematic depicting clustering of CDC RNA splice
junctions. FIG. 9 shows a linear representation of splice
acceptor/donor cluster sites relative to the canonical splice pair
AG/GU . . . AG. Acceptor/donor pairs are matched by their
respective color.
[0031] FIG. 10, comprising FIGS. 10A through 10E, depicts control
data for GFP fusion constructs. Control experiments we conducted
using the Olympus Fluoview FV1000 to illustrate the specificity of
the GFP fusion constructs.
[0032] FIG. 11, comprising FIGS. 11A and 11B, illustrates isolated
dendrite viability evaluation using mitochondrial function
measurement.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The morphology of a dendrite, and the manner of interaction
of a dendrite with surrounding structures, including other neurons
and neurites, including axons and other dendrites, plays a
significant role in the electrophysiological characteristics of the
dendrite. Dendritic interactions also play a role with respect to
the "connectedness" of a neuron within a synaptic network.
[0034] In the present invention, the presence of nuclear RNA
splicing machinery in dendrites is shown for the first time using
multiple localization procedures. It is also shown for the first
time herein that when isolated dendrites are transfected with a
.delta.-crystallin (CDC) pre-RNA or luciferase reporter RNA
containing an intron, splicing at canonical and cryptic splice
acceptor/donor sites is obtained. Additionally, in vitro
synaptoneurosome experiments set forth herein for the first time
show that the cytoplasmic subcellular fraction contains a similar
complement of splicing constituents that is able to splice CDC
pre-RNA. Analysis of the CDC pre-mRNA spliced RNAs reveals that a
subset of the dendritically-spliced transcripts can be locally
translated.
[0035] Therefore, the present application features methods for
dendritic-local splicing of RNA. This is because, as shown herein
for the first time, RNA splicing can occur extranuclearly, and in
particular, in the cytoplasm of a cell. The invention also features
methods of remodeling the electrochemical and the physical
structure of a dendrite using methods related to dendrite-local RNA
splicing. Further, the invention features methods of remodeling the
electrochemical and the physical structure of a dendrite using
methods related to dendrite-local RNA splicing and subsequent
dendrite-local protein translation.
[0036] The present invention also features methods of remodeling
the electrochemical and the physical structure of a dendrite using
methods related to dendrite-local RNA splicing any mammalian cell
cytoplasm or non-nuclear cellular compartment or fraction, as well
as methods of remodeling the electrochemical and the physical
structure of a dendrite using methods related to dendrite-local RNA
splicing and subsequent dendrite-local protein translation in any
mammalian cell cytoplasm or non-nuclear cellular compartment or
fraction.
[0037] It is also a feature of the present invention to provide
methods for the cytoplasmic splicing of RNA in dendrites. Further,
the invention provides methods cytoplasmic splicing and translation
of RNA in any mammalian cell cytoplasm or non-nuclear cellular
compartment or fraction.
DEFINITIONS
[0038] Unless defined otherwise, all technical and scientific terms
used herein generally have the same meaning as commonly understood
by one of ordinary skill in the art to which this invention
belongs. Generally, the nomenclature used herein and the laboratory
procedures in cell culture, molecular genetics, organic chemistry,
and nucleic acid chemistry and hybridization are those well known
and commonly employed in the art.
[0039] Standard techniques are used for nucleic acid and peptide
synthesis. The techniques and procedures are generally performed
according to conventional methods in the art and various general
references (e.g., Sambrook and Russell, 2001, Molecular Cloning, A
Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor,
N.Y., and Ausubel et al., 2002, Current Protocols in Molecular
Biology, John Wiley & Sons, NY), which are provided throughout
this document.
[0040] The nomenclature used herein and the laboratory procedures
used in analytical chemistry and organic syntheses described below
are those well known and commonly employed in the art. Standard
techniques or modifications thereof, are used for chemical
syntheses and chemical analyses.
[0041] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0042] As used herein, amino acids are represented by the full name
thereof, by the three letter code corresponding thereto, or by the
one-letter code corresponding thereto, as indicated in the
following table: TABLE-US-00001 Full Name Three-Letter Code
One-Letter Code Aspartic Acid Asp D Glutamic Acid Glu E Lysine Lys
K Arginine Arg R Histidine His H Tyrosine Tyr Y Cysteine Cys C
Asparagine Asn N Glutamine Gln Q Serine Ser S Threonine Thr T
Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L Isoleucine
Ile I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan
Trp W
[0043] As used herein, to "alleviate" a disease, disorder or
condition means reducing the severity of one or more symptoms of
the disease, disorder or condition.
[0044] An "isolated nucleic acid" refers to a nucleic acid segment
or fragment which has been separated from sequences which flank it
in a naturally occurring state, e.g., a DNA fragment which has been
removed from the sequences which are normally adjacent to the
fragment, e.g., the sequences adjacent to the fragment in a genome
in which it naturally occurs. The term also applies to nucleic
acids which have been substantially purified from other components
which naturally accompany the nucleic acid, e.g., RNA or DNA or
proteins, which naturally accompany it in the cell. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (e.g., as a cDNA
or a genomic or cDNA fragment produced by PCR or restriction enzyme
digestion) independent of other sequences. It also includes a
recombinant DNA which is part of a hybrid gene encoding additional
polypeptide sequence.
[0045] In the context of the present invention, the following
abbreviations for the commonly occurring nucleic acid bases are
used. "A" refers to adenosine, "C" refers to cytidine, "G" refers
to guanosine, "T" refers to thymidine, and "U" refers to
uridine.
[0046] A "polynucleotide" means a single strand or parallel and
anti-parallel strands of a nucleic acid. Thus, a polynucleotide may
be either a single-stranded or a double-stranded nucleic acid.
[0047] The term "nucleic acid" typically refers to large
polynucleotides.
[0048] The term "oligonucleotide" typically refers to short
polynucleotides, generally, no greater than about 50 nucleotides.
It will be understood that when a nucleotide sequence is
represented by a DNA sequence (i.e., A, T, G, C), this also
includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces
"T."
[0049] Conventional notation is used herein to describe
polynucleotide sequences: the left-hand end of a single-stranded
polynucleotide sequence is the 5'-end; the left-hand direction of a
double-stranded polynucleotide sequence is referred to as the
5'-direction.
[0050] The direction of 5' to 3' addition of nucleotides to nascent
RNA transcripts is referred to as the transcription direction. The
DNA strand having the same sequence as an mRNA is referred to as
the "coding strand"; sequences on the DNA strand which are located
5' to a reference point on the DNA are referred to as "upstream
sequences"; sequences on the DNA strand which are 3' to a reference
point on the DNA are referred to as "downstream sequences."
[0051] A "portion" of a polynucleotide means at least at least
about twenty sequential nucleotide residues of the polynucleotide.
It is understood that a portion of a polynucleotide may include
every nucleotide residue of the polynucleotide.
[0052] A "recombinant polypeptide" is one which is produced upon
expression of a recombinant polynucleotide.
[0053] "Polypeptide" refers to a polymer composed of amino acid
residues, related naturally occurring structural variants, and
synthetic non-naturally occurring analogs thereof linked via
peptide bonds, related naturally occurring structural variants, and
synthetic non-naturally occurring analogs thereof. Synthetic
polypeptides can be synthesized, for example, using an automated
polypeptide synthesizer.
[0054] The term "protein" typically refers to large
polypeptides.
[0055] The term "peptide" typically refers to short
polypeptides.
[0056] Conventional notation is used herein to portray polypeptide
sequences: the left-hand end of a polypeptide sequence is the
amino-terminus; the right-hand end of a polypeptide sequence is the
carboxyl-terminus.
[0057] As used herein, to "treat" means reducing the frequency with
which symptoms of a disease, disorder, or adverse condition, and
the like, are experienced by a patient.
[0058] As the term is used herein, "modulation" of a biological
process refers to the alteration of the normal course of the
biological process.
[0059] As used herein, the term "remodel" relates to an alteration
of the state or condition of something from a previous state or
condition. For example, a neural network is "remodeled" as a result
of a procedure or treatment if at least one neural connection or
interface is changed from a previous state or condition as a result
of the procedure or treatment.
[0060] The term "dendrite contact," as used herein, indicates
physical contact of a dendrite with another physiological
structure, including, but not limited to a second dendrite, an
axon, a neurite, or a soma. The term "dendrite interaction" or
"interaction with a dendrite" indicates at least one of chemical
and physical contact of a dendrite with another physiological
structure, but does not require physical contact with a
dendrite.
[0061] As used herein, the term "synaptic network" refers to an
interconnected network of neurons, and may include other
components. The term "neural network" also refers to a network of
neurons, and may include other components.
Description of the Invention
A. Methods of Splicing and Translating RNA
[0062] The present invention features a method of splicing RNA in a
dendrite. It has been shown for the first time herein that, using a
dendrite, which is a specialized extension of a neuron involved in
interconnecting other neurons, RNA can be spliced outside of the
nucleus. It has also been shown for the first time herein that,
using the splicing capability of dendrites, RNA can be spliced
outside of the nucleus. RNA splicing is useful for various purposes
in the field of genetics and molecular biology, including the
production of stable and properly organized transcripts, as well as
the production of correctly-spliced and functional translated gene
products. RNA splicing as described in the present invention is
also useful for the genetic treatment of diseases in which the
proper transcript is unstable or reactive. Delivery of a stable,
unspliced transcript to a cell or an organism can enable the
subsequent splicing and translation of the desired transcript.
[0063] In one embodiment of the invention, a method of splicing an
RNA includes transfecting a dendrite with an RNA comprising at
least one intron, wherein the dendrite comprises at least one
component of a spliceosome, and the component of a spliceosome is
capable of splicing an RNA. The transfected RNA is spliced by the
spliceosome component within the dendrite. In one aspect of the
invention, the dendrite is an isolated dendrite. That is, the
dendrite is isolated from the rest of a neuron.
[0064] In an aspect of the invention, an RNA splicing
donor/acceptor pair is a canonical pair. In another aspect of the
invention, an RNA splicing donor/acceptor pair is an atypical pair.
In yet another aspect of the invention, an RNA splicing
donor/acceptor pair is a cryptic pair.
[0065] In an embodiment of the invention, an RNA comprising at
least one intron is a pre-RNA. In one aspect, the RNA is a
pre-mRNA. As discussed elsewhere herein, a pre-RNA is any RNA that
can be processed to give rise to a molecularly distinct RNA
molecule. Examples of pre-RNAs include, but are not limited to, an
RNA that can be spliced to produce an RNA splice product that is
shorter in length. As will be understood by the skilled artisan,
intron-containing RNA useful in the present invention can be
prepared in any number of ways, and the method of preparation of
RNA should not be considered limiting. By way of a non-limiting
example, RNA useful in the present invention may be prepared by
methods including isolation of native RNA from a cell, isolation of
RNA from a recombinant system in which a recombinant DNA construct
was used to transcribe RNA, or from an RNA virus or a recombinant
RNA virus (eg., rhinovirus, hepatitis C).
[0066] In one embodiment of the invention, RNA comprising at least
one intron is derived from a recombinant DNA construct. A construct
useful in the present invention is designed to provide a
transcribed RNA comprising at least one splice site. Constructs
useful in the present invention include, but are not limited to,
pEGFP-N1, pDsRed-N1, SF1/mBBP-GFP, U2AF65-GFP, a GFP construct, a
DsRed construct, a histone 2B-YFP construct. In another embodiment
of the invention, a construct can also be engineered to comprise at
least one intron. In one aspect, a construct is engineered so that
RNA splicing produces a detectable product. By way of a
non-limiting example, a construct can be engineered such that
splicing results in an output of fluorescence.
[0067] In an embodiment of the invention, at least one spliceosome
component is present in a cytoplasm useful for splicing an RNA
according to a method of the present invention. In one aspect, a
spliceosome component is identified by a detection process, as
described in detail in the Experimental Examples of the present
application. In another aspect, a spliceosome component can be
recombinantly engineered to exist in a cytoplasm useful for
splicing an RNA according to a method of the present invention.
Such recombinant techniques are well-known in the art, and will not
be discussed in detail herein. For example, the invention
encompasses expression vectors and methods for the introduction of
exogenous DNA into cells with concomitant expression of the
exogenous DNA in the cells such as those described, for example, in
Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York), and in Ausubel et al. (1997,
Current Protocols in Molecular Biology, John Wiley & Sons, New
York).
[0068] By way of a non-limiting example, spliceosome components
useful in the present invention include, but are not limited to,
Y14, Magoh, RNPS1, SC-35, SF2, U2AF65, Smith antigen, pan-SR
antigen, U1 snRNP, U2 snRNP, U4 snRNP, U5 snRNP and U6 snRNP.
[0069] In another embodiment of the invention, a dendrite is a
component of a neuron. That is, the dendrite is attached to a
neuron comprising at least a soma. In one embodiment, the neuron is
isolated. In another embodiment, the neuron is maintained in
culture. In yet another aspect of the invention, a dendrite is a
component of a neuron, wherein the neuron is in vivo, in a living
organism. In one embodiment, the neuron is a part of a neural
network. In another embodiment, the neuron is a transplanted
neuron. In one aspect, the neuron is a transplanted neuron that is
not a part of a neural network, but has the potential to be
stimulated to integrate into an existing neural network.
[0070] This is because it has been shown herein for the first time
that dendrites contain components of the RNA spliceosome and of the
cellular translation machinery, and that RNA can be spliced and
subsequently translated outside of the nucleus. In one embodiment,
the present invention features a method of splicing an
intron-containing RNA in the cytoplasm of a dendrite, followed by
the translation of the spliced RNA to the corresponding
protein.
[0071] In one embodiment, a method of translating a spliced RNA
includes transfecting a dendrite with an RNA comprising at least
one intron, wherein the dendrite comprises at least one component
of a spliceosome, and the component of a spliceosome is capable of
splicing an RNA. The transfected RNA is spliced by the spliceosome
component within the dendrite, and the spliced RNA is translated by
the components of the tranaslation machinery located within the
dendrite.
[0072] In another embodiment of the invention, a method of
translating a spliced RNA involves an isolated synaptoneurosome. In
an embodiment of the invention, a method of translating a spliced
RNA includes contacting a synaptoneurosome with an RNA comprising
at least one intron, wherein the synaptoneurosome comprises at
least one component of a spliceosome, and the component of a
spliceosome is capable of splicing an RNA. The transfected RNA is
spliced by the spliceosome component within the synaptoneurosome,
and the spliced RNA is translated by the components of the
tranaslation machinery located within the synaptoneurosome.
[0073] In an aspect of the invention, the dendrite in which an RNA
is translated is an isolated dendrite. In another aspect of the
invention, a dendrite in which an RNA is translated is a component
of a neuron. In one embodiment, the neuron is isolated. In another
embodiment, the neuron is maintained in culture. In yet another
aspect of the invention, a dendrite in which an RNA is translated
is a component of a neuron, wherein the neuron is in vivo, in a
living organism. In one embodiment, the neuron is a part of a
neural network. In another embodiment, the neuron is a transplanted
neuron. In one aspect, the neuron is a transplanted neuron that is
not a part of a neural network, but has the potential to be
stimulated to integrate into an existing neural network.
[0074] This is further because, as described in detail elsewhere
herein, an RNA can be spliced and translated extranuclearly, within
a dendrite. Translation of a particular spliced transcript locally
within a dendrite can be used to remodel the dendrite. That is, the
administration of a specific intron-containing RNA to a dendrite
can be used to direct the local production of a protein that can be
used to effect a change in the structure, biology, or
electrophysiochemistry of a dendrite. By way of a non-limiting
example, an intron-containing RNA is transfected into a dendrite,
whereby the transfected RNA is specifically spliced, and the
spliced transcript is translated locally to produce a protein that
is useful for altering the physical structure and arrangement of
the dendrite, thereby altering the neural connections of the
dendrite. Such proteins include, but are not limited to, cadherin,
BDNF receptor, and neurexin.
[0075] Therefore, the present invention also features a method of
remodeling a dendrite, wherein the method includes transfecting a
dendrite with an RNA comprising at least one intron, wherein the
dendrite comprises at least one component of a spliceosome, and the
component of a spliceosome is capable of splicing an RNA. The
transfected RNA is spliced by the spliceosome component within the
dendrite, and the spliced RNA is translated by the components of
the tranaslation machinery located within the dendrite. The protein
produced by the translation consequently effects remodeling of the
dendrite. In one aspect of the invention, the dendrite remodeling
includes a physical alteration or restructuring of the dendrite. In
another aspect of the invention, the dendrite remodeling includes
an alteration of the chemical nature of the dendrite. In yet
another aspect of the invention, the dendrite remodeling includes
an alteration of the electrophysiochemical nature of the dendrite.
In still another aspect of the invention, the dendrite remodeling
includes an alteration of more than one property of the dendrite,
such as, but not limited to, a physical restructuring of the
dendrite that subsequently results in an alteration of the
biological properties of the dendrite.
[0076] RNAs encoding proteins useful for remodeling a dendrite or a
synaptic network include, but are not limited to, RNAs encoding
cadherin, neurexin, synaptophysin, tubulin, microtubule associated
proteins, and actin. As will be understood by the skilled artisan,
when armed with the present application, RNAs encoding any protein
known to be involved in the growth, homeostasis or remodeling of a
dendrite are useful in the present invention. As will be understood
by the skilled artisan, such RNAs may be pre-RNAs, and may be
spliced to form the final useful RNA molecule.
[0077] One of skill in the art will understand, when armed with the
present disclosure, that a multitude of properties of a dendrite,
and by association, of a neuron, can be affected by the methods of
the present invention. While not wishing to be bound by any
particular theory, one of skill in the art will understand that a
method of the present invention is useful for the growth of a
synaptic network, by way of dendrite stimulation and remodeling, to
form new and additional neuronal connections through dendrite
remodeling. As will be understood by the disclosure set forth
herein, such neural network growth and/or remodeling is useful in
vitro, in a neuronal cell culture, or in vivo, in a patient in need
of neural network growth and/or remodeling. Conditions for which
neural network remodeling is useful include, but are not limited
to, neurodegenerative diseases, such as Parkinson's disease,
Alzheimers disease, Huntington's disease, fragile X disease, Downs'
syndrome, and neuropsychiatric illnesses such as depression,
schizophrenia, and schizo-affective disorders.
[0078] Therefore, the present invention also features a method of
remodeling a synaptic network, wherein the method includes
transfecting a dendrite with an RNA comprising at least one intron,
wherein the dendrite comprises at least one component of a
spliceosome, and the component of a spliceosome is capable of
splicing an RNA. The transfected RNA is spliced by the spliceosome
component within the dendrite, and the spliced RNA is translated by
the components of the translation machinery located within the
dendrite. The protein produced by the translation consequently
effects remodeling of the dendrite, which results in remodeling of
a synaptic network. In particular, the synaptic network comprising
interaction with the transfected dendrite will thereby be
remodeled.
[0079] As set forth in greater detail elsewhere herein, dendrite
remodeling, and therefore, remodeling of the synaptic network
comprising interaction with the transfected dendrite, includes one
or more of a physical alteration or restructuring of the dendrite,
alteration of the chemical nature of the dendrite, alteration of
the electrophysiochemical nature of the dendrite, and alteration of
the biological properties of the dendrite.
[0080] While the present invention is described in relation to the
extranuclear, cytoplasmic splicing and translation of an RNA in
relation to a dendrite, the skilled artisan reading the present
disclosure will understand that the present invention is equally
applicable to any cell. That is, the skilled artisan will
understand that the methods of the present invention are equally
applicable to use in the cytoplasm of any mammalian cell,
including, but not limited to, human, primate, mouse, rat, equine,
sheep, goat, pig and dog, among others. Further, the skilled
artisan will understand that the methods of the present invention
are based upon the phenomenon of RNA splicing, and subsequent
translation of the spliced RNA, which processes are essential
components of all mammalian cells.
[0081] Based on the disclosure set forth herein, the skilled
artisan will understand how to identify components of the
spliceosome extranuclearly, in the cytoplasm, and further, will
understand how to identify components of the translation machinery
in the cytoplasm. Further still, the skilled artisan, when equipped
with the disclosure of the present invention, will understand how
to assay for extranuclear RNA splicing and for translation of the
spliced RNA product. Based on the extensive disclosure set forth
herein, the routineer will understand that the present disclosure
guides the skilled artisan to assay for extranuclear RNA splicing
and for translation of the spliced RNA product using techniques
available in the art and within the realm of ordinary and routine
experimentation.
[0082] Therefore, the present invention also includes a method of
splicing an RNA, including transfecting a cell with an RNA
comprising at least one intron, wherein the cell comprises at least
one component of a spliceosome in the cytoplasm, and the component
of a spliceosome is capable of splicing an RNA. The transfected RNA
is spliced by the spliceosome component within the cytoplasm of the
cell. In one aspect of the invention, the cell is an isolated cell.
As will be understood based on the discussion set forth herein, the
cell can be any mammalian cell comprising a cytoplasm.
[0083] Further, in an embodiment of the invention, a method of
translating a spliced RNA includes transfecting a cell with an RNA
comprising at least one intron, wherein the cell comprises at least
one component of a spliceosome in the cytoplasm, and the component
of a spliceosome is capable of splicing an RNA. The transfected RNA
is spliced by the spliceosome component within the cytoplasm, and
the spliced RNA is translated by the cell. In another embodiment,
the spliced RNA is translated by the components of the translation
machinery located within the cytoplasm. In one aspect of the
invention, the cell in which a spliced RNA is translated is an
isolated cell. In another aspect of the invention, a cell in which
a spliced RNA is translated is a component of a cell culture. In
yet another aspect, a cell in which a spliced RNA is translated is
part of a living organism, including, but not limited to, a
human.
B. Methods of Assaying for the Presence of Spliceosome Components
in the Cytoplasm of a Cell
[0084] The present invention further includes a method of
identifying a functional complement of spliceosome components in a
cell. In one embodiment, a method of identifying a functional
complement of spliceosome components in a cell comprises the
introduction of an RNA, comprising at least one intron, into a
non-nuclear compartment of a cell or of a non-nuclear sub-cellular
fraction. In one aspect, the method comprises the step of detection
of any splice products resulting from the administration of an RNA.
In another aspect, the method comprises the step of detection of
any protein produced as a result of the production of splice
products resulting from the administration of an RNA.
[0085] By way of a non-limiting example, a cryptic splice site in
exon 7 of the ElaPDH gene binds with high affinity to SC35 of the
spliceosome machinery (Gabut et al., 2005, MCB 25:3286-3294). A
construct that contains this splice site can be used according to a
method of the present invention to produce a splice product that
can be detected in a non-nuclear compartment of a cell or in a
non-nuclear sub-cellular fraction.
EXPERIMENTAL EXAMPLES
[0086] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
Materials and Methods
[0087] Immunohistochemical detection of splicing proteins in
neurons: E18 rat hippocampal neurons were prepared as previously
described. 8-10 days later, cultured neurons were washed in
pre-warmed Hank's balanced salt solution supplemented with 850
mg/Liter sodium bicarbonate, 20 mM HEPES, pH 7.4, and 1 mM sodium
pyruvate then 1.times.PBS/0.12 M sucrose for one minute each. Cells
were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in
PBS/0.12 M sucrose for 7 minutes and washed three times in PBS/0.12
M sucrose and two times in PBS/5mM MgCl2 for 5 minutes each. Cells
were then permeabilized with 0.3% Triton-X 100/PBS for 5 minutes,
washed three times in PBS/5 mM MgCl2 for 5 minutes each, and stored
in blocking solution (10% goat serum (Sigma), 0.1% fish gelatin,
0.1% Tween 20, in PBS] for 2 hrs. Cells were incubated overnight at
4.degree. C. with the following primary antibodies diluted in 1%
blocking solution/PBST(0.1% Tween/PBS): anti-Sm proteins (5
.mu.g/ml, Lab Vision Corporation), anti SF2 (10 .mu.g/ml, Zymed
Laboratories INC. (this antibody does not recognize SC-35 or SF2)),
anti-SC-35 (1:500, Accurate Chemical and Scientific Corporation),
anti-SR (10 ug/ml Zymed Laboratories INC) and anti-U2AF65(10 ug/ml
Zymed Laboratories INC). Cells were then washed 3 times in PBST 5
minutes each and incubated for 2 hours in goat anti-mouse Qdot 525
(1:50, Quantum Dots, QDot Corp.). Neurons were washed 3 times in
PBST for 10 minutes each, then sequentially labeled with rabbit
polyclonal anti-MAP2 antibody (1:4000, kindly provided by Craig
Garner, Stanford University) and a second primary antibody in 1%
blocking solution/PBST overnight at 4.degree. C. then washed 3
times in PBS/Tween and incubated 2 hours in Cy5 secondary antibody
(1:250) and Qdot 605 (Quantum Dot 1:50). Following incubation,
cells were washed 3 times in PBST, mounted with vectashield, and
coverslips sealed with nail polish. Antibody dilution series were
done with each antibody to determine the optimal dilution to use
for these studies and secondary antibodies, absent primary
antibodies showed no specific binding (data not shown). Confocal
imaging was performed using a Fluoview 1000 confocal microscope
(Olympus) and a three-channel confocal microscope (Prairie
Technologies, WI) attached to an Olympus BX50 fixed-stage upright
microscope, with excitation at 458 nm and 633 nm for imaging
quantum dot (525,605) and Alexa 633, respectively. For selective
emission collection, 515.+-.15 nm and 585.+-.20 nm band pass
filters were used for quantum dot imaging. To prevent bleed
through, each quantum dot was imaged to setup laser power and
photomultiplier gain before imaging triple-labeled samples. To
confirm a lack of bleed-through between channels and confirm
specificity of reactivity, samples were imaged in parallel when
primary antibody was omitted. In some experiments we collected the
emission spectra and confirmed the identity of each label. Z-stacks
of confocal images were acquired by incrementing each image by 0.2
.mu.m. Images were processed using Metamorph software (Universal
Imaging, West Chester, Pa.). Other immunofluorescent images were
taken on a Zeiss Axiovert 200 microscope attached to an Orca-ER
camera (Hamamatsu) and processed with Axiovision 3.1 software
(Zeiss), an Olympus IX81 microscope using FluoView, or an Olympus
IX71 microscope using an Olympus DP12 camera.
[0088] Generation of spliceosome-related RBP constructs: cDNAs
encoding rat Magoh, RNPS1, SF1/mBBP, USAF65, UAP56, and Y14 were
isolated from whole rat brain by reverse transcription polymerase
chain reaction using Pfu Turbo (Stratagene). Primers were designed
according to the previously reported mouse or human sequences in
GenBank. Primers for reIF4A3 are
5'-AATGAATTCGCCACCATGGCGGCTAACGCCACGATGGCG-3' (SEQ ID NO: 1)
(sense; underlined nucleotides depict EcoRI site) and
5'-ATTTGGATCCCGAATTAGGTCAGCCACATTCATGGG-3' (SEQ ID NO:2)
(antisense; underlined nucleotides depict BamHI site). Primers for
rMagoh are 5'-AATAAGCTTGCCACCATGGAGAGTGACTTTTACCTGCGT -3' (SEQ ID
NO:3) (sense; underlined nucleotides depict HinDIII site) and
5'-ATTGACCGGTGGGATTGGTTTAATCTTGAAGTGTAA-3' (SEQ ID NO:4)
(antisense; underlined nucleotides depict AgeI site). Primers for
rRNPSI are 5'-AATAAGCTTGCCACCATGGATTTATCAGGAGTGAAAAAG-3' (SEQ ID
NO:5) (sense; underlined nucleotidesdepict HindDIII site) and
5'-ATTGACCGGTGGGAGCAGCCGTGAACCAACAGT-3' (SEQ ID NO:6) (antisense;
underlined nucleotides depict AgeI site). Primers for rSF1/mBBP are
5'-AATGCTAGCGCCACCATGGCGACCGGAGCGAACGCCACG-3' (SEQ ID NO:7) (sense;
underlined nucleotides depict NheI site) and
5'-ATTTGGATCCCAATGGGCGCGGAAAGTCCTCAC-3' (SEQ ID NO:8) (antisense;
underlined nucleotides depict BamHI site). Primers for rU2AF65 are
5'-AATAAGCTTGCCACCATGGACTTCTTCAACGCCCAGATG-3' (SEQ ID NO:9) (sense;
underlined nucleotides depict HinDIII site) and
5'-ATTTGGATCCCAGAAGTCCCGACGGTGGTACGA-3' (SEQ ID NO: 10) (antisense;
underlined nucleotides depict BamHI site). Primers for rUAP56 are
5'-AATAAGCTTGCCACCATGGCAGAGAACGATGTGGACAAT-3' (SEQ ID NO:11)
(sense; underlined nucleotides depict HinDIII site) and
5'-ATTTGGATCCCGTGTCTGTTCAATGTAGGAGGA (SEQ ID NO:12) (antisense;
underlined nucleotides depict BamHI site). Primers for rY14 are
5'-AATAAGCTTGCCACCATGGCGGACGTGCTGGATCTTCAC-3' (SEQ ID NO: 13)
(sense; underlined nucleotides depict HinDIII site) and
5'-ATTTGGATCCCGACGGCGTCTCCGGTCTGGACTCCT-3' (SEQ ID NO:14)
(antisense; underlined nucleotides depict BamHI site). The PCR
products were digested with the appropriate restriction enzyme and
inserted into pEGFP-N1 or pDsRed-N1 (Clontech). All sequences were
verified by sequencing and contain the full coding region in-frame
with either GFP or DsRed.
[0089] DNA/Ca2+-phosphate coprecipitation transfection of DNA
constructs into primary neuronal cell cultures: Primary hippocampal
neurons were cultured for 7-10 days in vitro and then transfected
as described by Park et al. with minor modifications (Park et al.,
2004, Neurosci. Lett. 361:220-4 [36]).
[0090] In situ hybridization of U1 RNA: Hippocampal neurons were
fixed in 4% paraformamide (10 min), washed 3.times.5 min in 0.01%
triton-X100 PBS (PBST), washed 7 min in 0.2% triton-x100, PBS, and
washed 2.times.5 min in PBS. After 3 hours in prehybridization
buffer, at 50.degree. C., hybridization buffer (50% formamide,
5.times.SSC, 1.times. Denhart's, 8% Dextran Sulfate, 10 mM DTT,
yeast tRNA (50 .mu.g/ml)) with 5 ng probe/.mu.l and 8% dextran
sulfate was added at 50.degree. C. overnight. These hybridization
conditions are quite stringent. Cells were washed 2.times.5 min
2.times.SSC at 50.degree. C., 2.times.10 min 0.22.times.SSC at RT,
and 1.times.30 min PBS1 .mu.g/ml RNase A. Cells were then washed
2.times.5 min in PBS, blocked in PBST+10% goat serum for 30 min.
Digoxigenin-labeled riboprobe antisense to bases 20 to 70 of U1 rat
snRNA or GAD65 mRNA were detected with alkaline-phosphatase
conjugated anti-DIG antibodies and visualized with BCIP/NBT.
Antisense and RNase A controls were also performed.
[0091] CDC pre-RNA preparation and RNA transfection of neurons and
dendrites: Pre- or mature CDCRNAs (pcDNA3 plasmid construct
containing insert of exons 14 and 15 of the CDC gene either with or
lacking intermittent intron sequence and flanked with FLAG epitope;
these constructs were kindly provided by Gideon Dreyfuss) were
transcribed using Ambion's T7 mMessage mMachine kit. Approximately
1 .mu.g of RNA was mixed with 5 .mu.l of Geneporter (Gene Therapy
Systems) and incubated for 10 minutes at room temperature then
stored on ice until use. Isolated dendrites were prepared by
mechanical severing of the process from the cell soma and RNA/lipid
mixtures applied to dendrites as previously described. Following
transfection, (RS)-3,5-dihydroxy-phenylglycine (DHPG, Tocris) was
added to a final concentration of 20 .mu.M in order to stimulate
translation and isolated dendrites were incubated at 37.degree. C.
for one hour. Dendrites were washed four times in physiological
buffer and fixed in 4% paraformaldehyde/PBS for 20 minutes then
washed in PBS.
[0092] PCR Analysis of dendritic CDC RNA splice products: After
transfection of hippocampal neurons with CDC RNA, dendrites were
picked individually and a reverse transcription reaction performed
using primer `sp6`. An initial PCR reaction was performed using
primers sp6 and 5A, then analyzed by gel electrophoresis.
Successful amplification of mRNA results in the presence of a major
476 bp fragment, with spliced products. The area immediately below
the 476 bp band is excised and gel purified, then reamplified
utilizing the 5A primer and nested primer FLAG, then analyzed by
gel electrophoresis. Samples displaying a positive splicing
activity produce DNA fragments ranging in size from 125 bp to 476
bp. These fragments were gel purified, cloned and sequenced.
Reverse Transcription reaction: AMV-RT (Cape Cod) 42.degree. C. 30
min. DNA amplifications were performed with Accuprime PFX DNA
polymerase (Invitrogen): 1.times.95.degree. C. 2 min,
40.times.95.degree. C. 15 sec, 47.degree. C. 30 sec, 68.degree. C.
30 sec, 1.times.68.degree. C. 7:00 min. Gel extractions were
performed with the Qiaquick gel extraction kit (Qiagen). Fragments
were A-tailed with Amplitaq (Perkin-Elmer) 10 min 68.degree. C.,
then ligated to pGEM-T-Easy (Promega). Primers:
sp6-ATTTAGGTGACACTATAGA (SEQ ID NO: 15), FLAG-TTTATCGTCATCGTCTTTG
(SEQ ID NO: 16), 5A-CCAATCGATATACTTAGCC (SEQ ID NO: 17),
5B-GCCAGTGCCAAGCTTGCTGAC (SEQ ID NO: 18).
[0093] Synaptoneurosome preparation: Protocol is adapted from
(Weiler et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:7168-71; Rao
et al., 1993, J. Neurochem. 61:835-44 [49, 50]). Briefly, 6-8 week
old male Sprague-Dawley rats were sacrificed by cervical
dislocation followed by rapid decapitation. After removal of the
cerebellum, the brain is homogenized with a large dounce
homogenizer (Wheaton) in cold isolation medium containing: 320 mM
Sucrose, 10 mM Tris-HCl, 1 mM EDTA. The homogenate is spun at 3,500
rpm for three minutes and the supernatant is colleted and re-spun
at 10,000 rpm for ten minutes. After re-suspension of the resultant
pellet in 2.5 mls cold isolation medium, the solution is further
homogenized and mixed with 12% Ficoll (Sigma). 7% Ficoll and
isolation medium are then slowly layered on top of the homogenate,
and solutions are spun at 27 K RPM for 35 minutes (Beckman L8-55M).
Synaptoneurosome fractions are then collected between the 12% and
7% Ficoll layers and kept on ice. For Western blotting,
synaptoneurosomes or whole brain tissue is immediately pelleted and
lysed in the presence of protease inhibitors. Equal amounts of
protein (as determined by BradfordAssay) are run on NuPAGE 10%
Bis-Tris precut gels (Invitrogen), transferred to PVDF membrane
BioRad), stained using the antibodies listed above and visualized
using chemiluminescence (PerkinElmer). Beta-tubulin is used as a
loading control.
Experimental Example 1
Identification and Characterization of Splicesome Components in
Neuronal Dendrites.
[0094] The spliceosome, which catalyses the ATP dependent removal
of introns from nuclear pre-RNA, is a multi-megadalton complex of
proteins and small nuclear RNAs (snRNA) (Staley et al., 1998, Cell
92:315-26; Jurica et al., 2003, Mol. Cell 12:5-14). Even in the
nucleus, the distribution of pre-RNA splicing factors is not
uniform. Rather, with discrete sites of concentration and lower
levels of factors diffusely dispersed throughout the nucleoplasm,
speckles (splicing factor compartments) can be readily identified
with an antibody against the spliceosome assembly factor SC-35
(Lamond et al., 2003, Nat. Rev. Mol. Cell Biol. 4:605-12).
Initially, confocal microscopy was used to determine the
immunofluorescent localization of the non-nuclear splicing factor
domains (FIGS. 2A, 2D, and 2G) in primary neuronal dendrites.
Expression outside of the nucleus clearly was observed as a series
of puncta in the perinuclear space and dendrites (Buchhalter et
al., 1991, Brain Res. Bull. 26:333-8). It was subsequently
determined that other splicing factors associated with the
initiation and commitment steps of pre-RNA splicing were also
detectable in the dendritic arbor. Because SC-35 nuclear sites show
relatively limited amounts of uridine incorporation, speckles are
believed to be storage sites for numerous splicing factors and
serine/arginine (SR)-rich proteins (Moen et al., 1995, Hum. Mol.
Genet. 4:1779-89). Using antisera directed against four core
components of the pre-spliceosome (i.e. SF2, U2AF65, Smith antigen
(Sm), and pan-SR antigens), it was determined that these proteins
co-exist with dendritically localized SC-35. (Buchhalter et al.,
1991, Brain Res. Bull. 26:333-8) Each of these proteins has
previously been detected in speckles of somatic nuclei as well as
other nuclear subdomains.
[0095] Intrinsic to the pre-spliceosome complex are the Sm proteins
that combine with snRNAs to form the core constituents of the small
nuclear ribonucleoprotein particles (snRNPs); U1, U2, and U4-U6
(Will et al., 2001, Curr. Opin. Cell Biol. 13:290-301). In turn,
the U1 snRNP is recruited to the nascent pre-RNA via the
interaction between SF2 and intronic RNA sequence. During several
other transitions, U2AF65, SF1/mammalian branch point binding
protein (SF1/mBBP), and SR-rich proteins are subsequently recruited
to the branch point sequence embedded in pre-RNA introns wherein
rearrangements lock the U2 snRNP onto the pre-mRNA thereby
committing to nuclear pre-RNA splicing (Guth et al., 1999, Mol.
Cell Biol. 19:8263-71). When localization of these splicing factors
was examined, high expression of USAF65 was observed (FIG. 2B), Sm
antigen (FIG. 2E), SF2 (FIGS. 2H and 2K), and SR proteins (FIG. 2D)
in the nuclei of both neurons and glia. These data are consistent
with previous experiments noting their nuclear distribution.
However, in each experiment, small puncta, or granule-like
structures, were observed, located in proximal and distal portions
of dendrites and their branch points. These granule-like bodies
(Mattaj et al., 1985, Cell 40:111-8) were also observed in the
perinuclear space of both neurons and glia often juxtaposed with
the nuclear envelope (data not shown). For some proteins such as
SF2, more granule-like structures were observed in the perinuclear
space with reduced levels of SF2 puncta localized in proximal and
distal dendrites (Tacke et al., 1995, Embo. J. 14:3540-51). The
merged confocal images shown in FIGS. 2C, 2F, 2I and 2L illustrate
the co-localization of each of the antigens with speckles or with
each other. The presence of the pre-spliceosome-related antigens in
the dendritic domain of the neurons was confirmed by
co-localization of microtubule-associated protein 2 (MAP2)
immunofluorescence (inset FIG. 2A). MAP2 is a protein marker of the
somatodendritic domain of neurons. A predominantly nuclear
localization, in contrast, was observed when using an anti-histone
3 antibody (see FIGS. 10E and 10F).
[0096] Since the traditional pre-RNA splicing complex also employs
an RNA component, in situ hybridization (ISH) was performed for U1
RNA on rat hippocampal cultures. U1 RNA is the RNA component of the
U1 snRNP and is critical for initiating traditional RNA splicing.
ISH revealed the presence of U1RNA in the nucleus and more disperse
localization in the cytoplasm and dendrites of many neurons (FIG.
3A). U1 RNA was interspersed along the length of dendrites, with
noticeable staining occurring greater than 30 .mu.m from the
nucleus. Areas of moderate U1 staining also appeared at dendritic
branch points. Antisense competition controls, where an excess of
U1 antisense RNA is added to the prehybridization solution, washed
away and labeled U1 antisense RNA annealed, showed only slight
staining within the nucleus, potentially due to hybridization
within multiple copies of the U1 RNA genes in the genome (FIG. 3B).
Additionally, sense controls showed little staining. Dendrites are
identified by MAP2 immunostaining (FIGS. 3C and 3D). The appearance
of high levels of U1 RNA staining in the nucleus was expected
(Huang et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:305-8). By
way of comparison the presence of U1 RNA in dendrites is contrasted
with the dominant somatic localization of GAD65 mRNA in primary
hippocampal neurons (FIGS. 3E and 3F). These data showing U1 RNA
expression coupled with the evidence for pre-spliceosome
constituents dispersed throughout the dendritic cytoplasm indicate
that the assembly of functional core splicing components and more
complex splicing mRNPs which would be required for the processing
of select, dendritically localized mRNAs.
[0097] Following the initial commitment to splicing, constitutive
pre-mRNA processing is also accompanied by a series of dynamic
changes in the protein composition of the spliceosome. Three
individual subcomplexes of the spliceosome (i.e. A (previously
referred to as the pre-spliceosome), B*, and C) have recently been
purified and their components identified by mass spec analysis
(Hartmuth et al., 2002, Proc. Natl. Acad. Sci. U.S.A. 99:16719-24;
Jurica et al., 2002, Rna 8:426-39; Makarov et al., 2002, Science,
298:2205-8). Having already explored the distribution of
pre-spliceosome components, subcomplexes B* and C were examined.
Subcomplex B* temporally represents the mRNP remodeling just prior
to the first transesterification reaction while the catalytic C
subcomplex represents the splice-intermediate stage following this
first chemical step. In particular, spliced mRNAs acquire a set of
specific protein complexes that assemble near exon-exon junctions.
Components of this exon junction complex (EJC), a majority of which
are found in subcomplex C (Makarov et al., 2002, Science,
298:2205-8), include SRm160, RNPS1, UAP56, Aly/REF, Upf3, eIF4A3,
Y14, and Magoh (Kataoka et al., 2000, Mol. Cell 6:673-82[29]).
Assembling 20-24 nucleotides upstream of spliced junctions, these
proteins function in all manner of RNA metabolism including mRNA
export (Le Hir et al., 2000, Genes Dev. 14:1098-108), coactivation
of splicing (Blencowe et al., 1998, Genes Dev. 12:996-1009; Mayeda
et al., 1999, Embo. J. 18:4560-70; Mayeda et al. 1999, Mol. Cell
Biol. 19:1853-63), and nonsense-mediated mRNA decay (NMD) (Kim et
al., 2001, Science 293:1832-6). Well-characterized,
commercially-available antibodies, were used to determine the
dendritic localization of each of the splicing factors examined
herein. However, it was not always apparent that the antibodies
were able to detect the native proteins with sufficient sensitivity
using immunofluorescence. In part, this is likely due to the large
macromolecular mRNP complexes to which they are intrinsic that may
sterically hinder antibody access. To circumvent these difficulties
and as a secondary confirmation of splicing enzyme localization,
splicing RBPs were engineered, fused in-frame with GFP or DsRed at
the carboxy terminus so that the fluorescent marker would be
targeted to the subcellular sites where the splicing factors were
localized. The rat splicing factors were PCR cloned from rat brain
cDNA and their identities sequence verified. These splicing enzyme
sequences have been submitted to Genbank. For complex A-related
factors, SF1/mBBP-GFP were generated (FIGS. 4A and 4B), as were
U2AF65-GFP fusion contructs (FIGS. 4C and 4D). The U2AF65-GFP
construct was used as a secondary confirmation of
immunofluroescence data of splicing factor localization performed
in FIG. I (see also Table 1). For complex B*, a commercially
available antibody to the polypyrmidine tract-binding protein
associated splicing factor was used (PSF, FIGS. 4E-1 and 4F-1)
(Kanai et al., 2004, Neuron 43:513-25). Finally, for complex C, an
antibody to Aly/REF (FIGS. 4G-1 and 4H-1) and GFP or DsRed fusion
constructs containing the open reading frame of Magoh (FIGS. 4I and
4J), UAP56 (FIGS. 4L and 4M), Y14 (FIGS. 4N and 4O), and RNPS1 were
used. GFP or DsRed fusion constructs were transfected into primary
hippocampal cultures at 10-12 days in vitro using a calcium
phosphate protocol (Park et al., 2004, Neurosci. Lett. 361:220-4).
For low-resolution, whole cell images, the intensity of the laser
was increased to clearly show dendritic signal. In doing so, signal
in nuclei is often saturated wherein specific nuclear
subcompartment fluorescence is saturated. Photomontages of Z-stack
images illustrate the images obtained during these experiments.
SF1-mBBP-GFP and U2AF65-GFP showed consistent nuclear localization
complemented by larger puncta as well as more dispersed
granule-like structures throughout the perinuclear and dendritic
cytoplasm in higher magnification images. Of note is the similarity
in expression patterns obtained for U2AF65-GFP and the previous
U2AF65 immunofluorescence obtained in FIG. 2. Complex B* component
PSF showed an altogether separate pattern of localization. As
previously described, speckled nuclei were distinctly visible.
However, a low but significant cytoplasmic staining was visible in
the perinuclear space (see arrows in FIG. 4F-1). Experiments with
complex C cofactors showed some variation in expression depending
upon the protein. Aly/REF immunofluorescence was restricted mostly
to nuclei. However, a consistently low level of diffuse signal was
visible interspersed throughout the dendritic arbor. In comparison,
Magoh-GFP, Y14-GFP, UAP56-GFP, and RNPS1-GFP were scattered in
granule-like structures in the dendroplasm and speckle-like domains
in the nucleoplasm (see FIG. 10). Experiments performed with
anti-Magoh or-Y14 antibodies were used to confirm the pattern of
signal obtained with the Magoh- or Y14-GFP constructs. Magoh and
Y14 are known to directly interact and have been shown to be
important for oskar mRNA localization in Drosophila during
oogensis. When co-expressed in primary neurons, Magoh-GFP and
Y14-DsRed show nearly complete co-localization (FIG. 4K).
TABLE-US-00002 TABLE 1 Sequences of dendritically-spliced and
synaptoneurosome-spliced RNAs. EXON DONOR 5' INTRON 3' INTRON EXON
ACCEPTOR OVERLAP/NOTES CDC-preRNA Transfection of Isolated
Dendrites 1 TTAAGTGTTGTACAG ATACGAGAGCTCTAG CTTTGAGACACTAAC
GTGTCCCCAGGGAGC 0/D (SEQ ID NO:19) (SEQ ID NO:20) (SEQ ID NO:21)
(SEQ ID NO:22) 2 TCTCATTATTTAATG TGGTTGGAGGACACA CATGGAGAAGGCTCT
GACCCCTGAGTTGCT 0/C (SEQ ID NO:23) (SEQ ID NO:24) (SEQ ID NO:25)
(SEQ ID NO:26) 3 CTGTGCTCCAGGTTG CCACTGGAGTGATTT AGGAGAACATGGAGA
AGGCTCTGACCCCTG 0 (SEQ ID NO:27) (SEQ ID NO:28) (SEQ ID NO:29) (SEQ
ID NO:30) 4 TGCCACTGGAGTGAT TTCTACCCTCCAGGT CATGGAGAAGGCTCT
GACCCCTGAGTTGCT 0 (SEQ ID NO:31) (SEQ ID NO:32) (SEQ ID NO:33) (SEQ
ID NO:34) 5 AAGGTTTCAGCTTCT CATTATTTAATGTGG TCCTTTTGCAGGTCA
ACAAGGAGAACATGG 0 (SEQ ID NO:35) (SEQ ID NO:36) (SEQ ID NO:37) (SEQ
ID NO:38) 6 CGAGAGCTCTAGTCT GGTCCTAACATGAAG TGCAGGTCAACAAGG
AGAACATGGAGAAGG 0/D (SEQ ID NO:39) (SEQ ID NO:40) (SEQ ID NO:41)
(SEQ ID NO:42) 7 CATTATTTAATGTGG TTGGAGGACACATTT CTGCCTCTCCTCTTT
GCAGGTCAACAAGGA 0 (SEQ ID NO:43) (SEQ ID NO:44) (SEQ ID NO:45) (SEQ
ID NO:46) 8 TTGCGTGTTGTACAG ATACGAGAGCTCTAG CTTTGAGACACTAAC
GTGTCCCCAGGGAGC 0 (SEQ ID NO:47) (SEQ ID NO:48) (SEQ ID NO:49) (SEQ
ID NO:50) 9 CTGGAGTGATTTCTA CCCTCCAGGTAAGGT CTCTCTCTCTGCCTC
TCCTCTTTGCAGGTC 0 (SEQ ID NO:51) (SEQ ID NO:52) (SEQ ID NO:53) (SEQ
ID NO:54) 10 TTTCTACCCTCCAGG TAAGGTTTCAGCTTC CTCTTTGCAGGTCAA
CAAGGAGAACATGGA 0 (SEQ ID NO:55) (SEQ ID NO:56) (SEQ ID NO:57) (SEQ
ID NO:58) 11 TCTGGTCCTAACATG CTGACCCCTCACTCC AACATGGAGAAGGCT
CTGACCCCTGAGTTG 0 (SEQ ID NO:59) (SEQ ID NO:60) (SEQ ID NO:61) (SEQ
ID NO:62) 12 CCACTGGAGTGATTT CTACCCTCCAGGTAA CTTTGCAGGTCAACA
AGGAGAACATGGAGA 0 (SEQ ID NO:63) (SEQ ID NO:64) (SEQ ID NO:65) (SEQ
ID NO:66) 13 ACCTGCTGACTGCTG TGCTCCAGGTTGCCA CAAGGAGAACATGGA
GAAGGCTCTGACCCC 0 (SEQ ID NO:67) (SEQ ID NO:68) (SEQ ID NO:69) (SEQ
ID NO:70) 14 ATGTGGTTGGAGGAC CATTTTAAGTGTTGT CTCCTCTTTGCAGGT
CAACAACGAGAACAT 0 (SEQ ID NO:71) (SEQ ID NO:72) (SEQ ID NO:73) (SEQ
ID 140:74) 15 AGGTTGCCACTGGAG TGATTTCTACCCTCC GCCTCTCCTCTTTGC
AGGTCAACAAGGAGA 0 (SEQ ID NO:75) (SEQ ID NO:76) (SEQ ID NO:77) (SEQ
ID NO:78) 16 GTTTCAGCTTCTCAT TATTTAATGTGGTTG ATGGAGAAGGCTCTG
ACCCCTGAGTTGCTG 0 (SEQ ID NO:79) (SEQ ID N0:80) (SEQ ID NO:81) (SEQ
ID NO:82) 17 CTCTAGTCTGGTCCT AACATGAAGACTTGC AACAAGGAGAACATG
GAGAAGGCTCTGACC 0 (SEQ ID NO:83) (SEQ ID NO:84) (SEQ ID NO:85) (SEQ
ID NO:86) 18 CTGTGCTCCAGGTTG CCACTGGAGTGATTT CAAGGAGAACATGGA
GAAGGCTCTGACCCC 0 (SEQ ID NO:87) (SEQ ID NO:88) (SEQ ID NO:89) (SEQ
ID NO:90) 19 CTCATTATTTAATGT GGTTGGAGGACACAT AGAAGGCTCTGACCC
CTGAGTTGCTGTCTA 0 (SEQ ID NO:91) (SEQ ID NO:92) (SEQ ID NO:93) (SEQ
ID NO:94) 20 CTTCTCATTATTTAA TGTGGTTGGAGGACA GGAGAAGGCTCTGAC
CCCTGAGTTGCTGTC 0 (SEQ ID NO:95) (SEQ ID NO:96) (SEQ ID NO:97) (SEQ
ID NO:98) 21 AGTAAGGTTTCAGCT TCTCATTATTTAATGT GTTGTGTCTACTGAT
CGGGGTAGACTACAA 1 (SEQ ID NO:99) (SEQ ID NO:100) (SEQ ID NO:101)
(SEQ ID NO:102) 22 GACTGCTGTGCTCCA GGTTGCCACTGGAGT CTCTTTGCAGGTCAA
CAAGGAGAACATGGA 1 (SEQ ID NO:103) (SEQ ID NO:104) (SEQ ID NO:105)
(SEQ ID NO:106) 23 TGGAGTGATTTCTAC CCTCCAGGTAAGGTT GCAAATGATAACCTC
TCTCTCTGCCTCTCC 1 (SEQ ID NO:107) (SEQ ID NO:108) (SEQ ID NO:109)
(SEQ ID NO:110) 24 TAGTCTGGTCCTAAC ATGAAGACTTGCTCA GAGAAGGCTCTGACC
CCTGAGTTGCTGTCT 1 (SEQ ID NO:111) (SEQ ID NO:112) (SEQ ID NO:113)
(SEQ ID NO:114) 25 CATGAAGACTTGCTC ACTCCTACTGCTTGT CGAGAAGGCTCTGAC
CCCTGAGTTGCTGTC 1 (SEQ ID NO:115) (SEQ ID NO:116) (SEQ ID NO:117)
(SEQ ID NO:118) 26 GGACACATTTTAAGT GTTGTACAGATACCA TCTCTCTCTGCCTCT
CCTCTTTGCAGGTCA 1 (SEQ ID NO:119) (SEQ ID NO:120) (SEQ ID NO:121)
(SEQ ID NO:122) 27 GAGTGATTTCTACCC TCCAGGTAAGGTTTC GGAGAAGGCTCTGAC
CCCTGAGTTGCTGTC 1 (SEQ ID NO:123) (SEQ ID NO:124) (SEQ ID NO:125)
(SEQ ID NO:126) 28 AGGACACATTTTAAG TGTTGTACAGATACG AGGCTCTGACCCCTG
AGTTGCTGTCTACTG 1 (SEQ ID NO:127) (SEQ ID NO:128) (SEQ ID NO:129)
(SEQ ID NO:130) 29 TGCTGTGCTCCAGGT TGCCACTGGAGTGAT GACCCCTGAGTTGCT
GTCTACTGATCGGGT 1 (SEQ ID NO:131) (SEQ ID NO:132) (SEQ ID NO:133)
(SEQ ID NO:134) 30 GAGTGATTTCTACCC CCCTGTAAGGTTTCA AGAAGGCTCTGACCC
CCCTGAGTTGCTGTC 1 (SEQ ID NO:135) (SEQ ID NO:136) (SEQ ID NO:137)
(SEQ ID NO:138) 31 GTTGTACAGATACGA GCTCTAGTCTGGTCC AGGAGAACATGGAGA
AGGCTCTGACCCCTG 2 (SEQ ID NO:139) (SEQ ID NO:140) (SEQ ID NO:141)
(SEQ ID NO:142) 32 AGTAAGGTTTCAGCT TCTCATTATTTAATG CAAATGATAACCTCT
CTCTCTGCCTCTCCT 2 (SEQ ID NO:143) (SEQ ID NO:144) (SEQ ID NO:145)
(SEQ ID NO:146) 33 AGGTAAGGTTTCAGC CTCTCCTCTTTGCAG AACCTCTCTCTCTGC
CTCTCCTCTTTGCAG 2 (SEQ ID NO:147) (SEQ ID NO:148) (SEQ ID NO:149)
(SEQ ID NO:150) 34 TGACTGCTGTGCTCC CAGGTTGCCACTGGA GAGAAGGCTCTGACC
CCTGAGTTGCTGTCT 2 (SEQ ID NO:151) (SEQ ID NO:152) (SEQ ID NO:153)
(SEQ ID NO:154) 35 TTTCTACCCTCCAGG TAAGGTTTCAGCTTC AGAACATGGAGAAGG
CTCTGACCCCTGAGT 3/A (SEQ ID NO:155) (SEQ ID NO:156) (SEQ ID NO:157)
(SEQ ID NO:158) 36 TGCTGTGCTCCAGGT GCCACTGGAGTGATT GTCTACTGATCGGGT
AGACTACAAAGACGA 3 (SEQ ID NO:159) (SEQ ID NO:160) (SEQ ID NO:161)
(SEQ ID NO:162) 37 GATTTCTACCCCCCA TGTAAGGTTTCAGCT CCTTGATGAAGTCCA
TTCTTTGAGACACTA 3 (SEQ ID NO:163) (SEQ ID NO:164) (SEQ ID NO:165)
(SEQ ID NO:166) 38 GGTTTCAGCTTCTCA TTATTTAATGTGGTT CCTCTTTGCAGGTCA
ACAAGGAGAACATGG 3 (SEQ ID NO:167) (SEQ ID NO:168) (SEQ ID NO:169)
(SEQ ID NO:170) 39 CTCTAGTCTGGTCCT AACATGAAGACTTGC AAGGCTCTGACCCCT
GAGTTGCTGTCTACT 3 (SEQ ID NO:171) (SEQ ID NO:172) (SEQ ID NO:173)
(SEQ ID NO:174) 40 GCTGACTGCTGTGCT CCAGGTTGCCACTGG GACCCCTGAGTTGCT
GTCTACTGATCGGGT 4 (SEQ ID NO:175) (SEQ ID NO:176) (SEQ ID N0:177)
(SEQ ID NO:178) 41 GTGATTTCTACCCTC CAGGTAAGGTTTCAG CTCTCTCTCTGCCTC
TCCTCTTTGCAGGTC 4 (SEQ ID NO:179) (SEQ ID NO:180) (SEQ ID NO:181)
(SEQ ID NO:182) 42 TGATTTCTACCCTCC TGTAAGGTTTCAGCT TCTCTCTGCCTCTCC
TCTTTGCAGGTCAAC 4 (SEQ ID NO:183) (SEQ ID NO:184) (SEQ ID NO:185)
(SEQ ID NO:186) 43 TATTTAATGTGGTTG GAGGACACATTTTAA CTGACCCCTGAGTTG
CTGTCTACTGATCGG 4 (SEQ ID NO:187) (SEQ ID NO:188) (SEQ ID NO:189)
(SEQ ID NO:190) 44 GTGATTTCTACCCTC CAGGTAAGGTTTCAG TCTCTGCCTCTCCTC
TTTGCAGGTCAACAA 4/A (SEQ ID NO:191) (SEQ ID NO:192) (SEQ ID NO:193)
(SEQ ID NO:194) 45 CTGGTCCTAACATGA AGACTTGCTCACTCC GGGAGCAGCAAATGA
TAACCTCTCTCTCTG 4/D (SEQ ID NO:195) (SEQ ID NO:196) (SEQ ID NO:197)
(SEQ ID 14O:198) 46 ACCCTCCAGGTAAGG TTTCAGCTTCTCATT TGCAGGTCAACAAGG
AGAACATGGAGAAGG 4/A (SEQ ID NO:199) (SEQ ID NO:200) (SEQ ID NO:201)
(SEQ ID NO:202) 47 TTTAATGTGAAGACT TGCTCACTCCTACTG CTGATCGGGTAGACT
ACAAAGACGATGACC 5/D (SEQ ID NO:203) (SEQ ID NO:204) (SEQ ID NO:205)
(SEQ ID NO:206) 48 TGCTGTGCTCCAGGT TGCCACTGGAGTGAT CTCCTCTTTGCAGGT
CAACAAGGAGAACAT 5/A (SEQ ID NO:207) (SEQ ID NO:208) (SEQ ID NO:209)
(SEQ ID NO:210) 49 TCTGGTCCTAACATG AAGACTTGCGTGTTG AACAAGGAGAACATG
GAGAAGGCTCTGACC 6/B (SEQ ID NO:211) (SEQ ID NO:212) (SEQ ID NO:213)
(SEQ ID NO:214) 50 GTGCCAAGCTTGCTG ACTGCTGTGCTCCAG ACCCCTGAGTTGCTG
TCTACTGATCGGGTA 6 (SEQ ID NO:215) (SEQ ID NO:216) (SEQ ID NO:217)
(SEQ ID NO:218) 51 CTTGCTGACTGCTGT GCTCCAGGTTGCCAC CCCCTGAGTTGCTGT
CTACTGATCGGGTAG 6 (SEQ ID NO:219) (SEQ ID NO:220) (SEQ ID NO:221)
(SEQ ID NO:222) 52 TTGCTACTCCTACTG CTTGTTATGACCCCA AGTTGCTGTCTACTG
ATCGGGTAGACTACA 6 (SEQ ID NO:223) (SEQ ID NO:224) (SEQ ID NO:225)
(SEQ ID NO:226) 53 TCTGGTCCTAACATG AAGACTTGCTCACTC AACAAGGAGAACATG
GAGAAGGCTCTGACC 6/B (SEQ ID NO:227) (SEQ ID NO:228) (SEQ ID NO:229)
(SEQ ID NO:230) 54 TTTAATGTGAAGACT TGCTCACTCCTACTG CTGATCGGGTAGACT
ACAAAGACGATGACG (SEQ ID NO:231) (SEQ ID NO:232) (SEQ ID NO:233)
(SEQ ID NO:234) Luciferase-SV40 pre-RNA 1 AAAGTCCAAATTGTA
CCAAATTGTAAAATG TACTGTTTTTTCTTA CTCCACACAGGCATA 2 (SEQ ID NO:235)
(SEQ ID NO:236) (SEQ ID NO:237) (SEQ ID NO:238) 2 AAGTCCAAATTGTAA
AATGTAACTGTATTC CAGTTATAATCATAA CATACTGTTTTTTCT 2 (SEQ ID NO:239)
(SEQ ID NO:240) (SEQ ID NO:241) (SEQ ID NO:242)
3 TTACGTCGCCAGTCA AGTAACAACCGCGAA TAACAGTTATAATCA TAACATACTGTTTTT 3
(SEQ ID NO:243) (SEQ ID NO:244) (SEQ ID NO:245) (SEQ ID NO:246) 4
CTTACCGGAAAACTC GACGCAAGAAAAATC TGTTTTTTCTTACTC CACACAGGCATAGAG 4
(SEQ ID NO:247) (SEQ ID NO:248) (SEQ ID NO:249) (SEQ ID NO:250) 5
GATGACGGAAAAAGA GATCGTGGATTACGT CTCCTCCAAAAAAGA AGAGAAAGGTAGAAG 7
(SEQ ID NO:251) (SEQ ID NO:252) (SEQ ID NO:253) (SEQ ID NO:254)
Sequences derived from Synaptoneurosome Splicing of CDC pre-RNA 1
CTTGCTCACTCCTAC TGCTTGTTATGACCC CTACTGATCGGGTAG ACTACAAAGACGATG 0
(SEQ ID NO:255) (SEQ ID NO:256) (SEQ ID NO:257) (SEQ ID NO:258) 2
TTTTAAGTGTTGTAC AGATACGAGAGCTCT TCTCTGCCTCTCCTC GATAACCTCTCTCTC 0
(SEQ ID NO:259) (SEQ ID NO:260) (SEQ ID NO:261) (SEQ ID NO:262) 3
TTCAGCTTCTCATTA TTTAATGTGGTTGGA TCCTCTTTGCAGGTC AACAAGGAGAACATG 1
(SEQ ID NO:263) (SEQ ID NO:264) (SEQ ID NO:265) (SEQ ID NO:266) 4
AGGTTTCAGCTTCTC ATTATTTAATGTGGT CCTCTCTCTCTGCCT CTCCTCTTTGCAGGT 3
(SEQ ID NO:267) (SEQ ID NO:268) (SEQ ID NO:269) (SEQ ID NO:270) 5
CTGACTGCTGTGCTC CAGGTTGCCACTGGA CCCCACCACAGGCAG CTCAGATACACTTGG 3
(SEQ ID NO:271) (SEQ ID NO:272) (SEQ ID NO:273) (SEQ ID NO:274) 6
CCTCCAGGTAAGGTT TCAGCTTCTCATTAT TGCTGTCTACTGATC GGGTAGACTACAAAG 0
(SEQ ID NO:275) (SEQ ID NO:276) (SEQ ID NO:277) (SEQ ID NO:278) 7
AGGTTTCAGCTTCTC ATTATTTAATGTGGT AAATGATAACCTCTC TCTCTGCCTCTCCTC 0
(SEQ ID NO:279) (SEQ ID NO:280) (SEQ ID NO:281) (SEQ ID NO:282) 8
GCTTGTTATGACCCC ACCACAGGCAGCTCAG ACATGGAGAAGGCTC TGACCCCTGAGTTGC 7
(SEQ ID NO:283) (SEQ ID NO:284) (SEQ ID NO:285) (SEQ ID NO:286) 9
TTCAGCTTCTCATTA TTTAATGTGGTTGGA CATTCTTTGAGACAC TAACGTGTCCCCAGG 0
(SEQ ID NO:287) (SEQ ID NO:288) (SEQ ID NO:289) (SEQ ID NO:290) 10
CTTGCTGACTGCTGT GCTCCAGGTTGCCAC CTCTGACCCCTGAGT TGCTGTCTACTGATC 6
(SEQ ID NO:291) (SEQ ID NO:292) (SEQ ID NO:293) (SEQ ID NO:294)
NOTES A: AG/GU CONSENSUS; B: IDENTICAL SEQUENCE; C: FROM
TRANSFECTION/IVT EXPERIMENT; D: FROM TRANSLATION EXPERIMENTS
[0098] Three additional observations from these experiments are
noteworthy. First, when non-neuronal cells from the same cultures
are imaged to discern RBP-GFP expression, a distinct staining
pattern is routinely observed when compared to neurons that are
transfected on the same coverslip. For a majority of the RBP-GFP
constructs shown here (with the exception of U2AF65-GFP and
Magoh-GFP), subcellular distribution in glia is normally limited to
nuclei with extremely modest, if any at all, staining visible in
the cytoplasm (see FIG. 10 and FIGS. 2B-2E). Second, when neurons
are transfected with the pEGFP-N1 or pDsRed-N1 construct alone, two
types of expression are observed; neither of which are seen when we
have fused a splice factor RBP in frame with GFP or DsRed. In one
instance, GFP is seen distributed throughout the neuronal nuclei
and cytoplasm with equal intensity. Often it is not easy to discern
a separate nucleus in these GFP-expressing cells. In another
instance, the nuclei of neurons and glia have low diffuse levels of
GFP staining in nuclei that is offset with distinct cytoplasmic
staining and a visible perinuclear ring of expression (FIGS. 10F
and 10G). Finally, when we used a well-characterized histone 2B-YFP
construct (Platani et al., 2002, Nat. Cell. Biol. 4:502-8) we
observed a primarily nuclear pattern of expression (FIG. 10J).
These results suggest that specific splicing factors from each of
the spliceosome subcomplexes are distributed within the dendritic
cytoplasm of neurons, have a variant expression in non-neuronal
cells and may be capable of constitutively splicing pre-mRNAs into
mature transcripts. Furthermore, experiments conducted with the
histone 2B-YFP fusion construct suggests that there is no intrinsic
feature of the transfection techniques or the over-expressing GFP
fusion constructs that would predispose our experiments to
observing a dendritic localization.
Experimental Example 2
Amplification of Spliced CDC RNA from Isolated Dendrites.
[0099] Given the presence of splicing machinery in dendrites, the
potential for RNA splicing to occur in neuronal dendrites was
investigated. To perform these experiments, we utilized a pre-RNA
splicing construct comprised of exons 14 and 15 of the CDC gene
(Ohno et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:5187-91 [38])
to assess whether it can be spliced in isolated dendrites.
[0100] During the time course of these experiments, the viability
of isolated dendrites to splice pre-RNA transcripts was assesed.
Isolated dendrites have been previously shown to be translationally
active (Aakalu et al., 2001, Neuron 30:489-502; Crino et al., 1996,
Neuron 17:1173-87; Job et al., 2001, Proc. Natl. Acad. Sci. U.S.A
98:13037-42) with the additional ability to incorporate
post-translational sugar precursors of the secreted-protein
protein-glycosylation pathway (Torre et al., 1996, J. Neurosci.
16:5967-78). One very sensitive measure of dendritic compromise is
mitochondrial function. JC-1, a mitochondrial membrane potential
dye (Li et al., 2004, Cell 119:873-87; Arancia et al., 2004, Amino
Acids 26:273-82; Ogbourne et al., 2004, Cancer Res. 64:2833-9), was
added at varying time point to isolated dendrites. Sequestration of
JC-1 by functional mitochondria results in its polymerization and
the formation of fluorescent red aggregates (see FIGS. 3 and 10). A
comparison of the zero time point and the 5 hr time point show
similar levels of red fluorescence. Based on these criteria, these
isolated dendrites remain biochemically functional during the time
frame of these experiments.
[0101] In the CDC pre-RNA the 87 bp exon 14 and 73 bp exon 15 of
the chicken 8-crystalline gene are interrupted by a 257 bp intron.
The pre-RNA derived from this construct has been used for in vitro
pre-RNA splicing assays where spliceosome assembly and CDC pre-RNA
splicing were observed (Kataoka et al., 2000, Mol. Cell 6:673-82;
Ohno et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:5187-91).
Briefly, CDC pre-RNA was transcribed from the construct, encoated
with the polycationic lipid Metafectene, and manually applied onto
dendrites that were isolated from their cognate cell somas as
performed previously (Aakalu et al., 2001, Neuron 30:489-502; Crino
et al., 1996, Neuron 17:1173-87; Job et al., 2001, Proc. Natl.
Acad. Sci. U.S.A 98:13037-42; Kacharmina et al., 2000, Proc. Natl.
Acad. Sci. U.S.A. 97:11545-50). After incubation at 37.degree. C.
for 30 minutes, RNA was extracted from these isolated dendrites.
Reverse transcription of this RNA was primed with an SP6 directed
primer inherent to the transfected 3'-end of the CDC RNA construct.
The cDNA was then used as template in multiple rounds of PCR using
CDC-specific primers with nested primer sets. These PCR amplicons
were subcloned and sequenced to determine the splice boundaries
utilized in the dendritic subdomain of the neuron.
[0102] In addition to a large amount of unspliced CDC pre-RNA,
sequence analysis of 53 spliced CDC RNA sequences isolated from
eight independent experiments revealed that CDC pre-RNA was spliced
using both predicted, consensus and non-canonical splicing
sequences (FIG. 2). Four of these sequences adhere to the AG/GU
rule for nuclear splicing, while the other sequences make use of
cryptic splice sites (Lee et al. 1997, Cancer Res. 57:3131-4). Four
other sequences adhere to atypical AT-AC intron splicing, an event
requiring the incorporation of SR proteins (Hastings et al., 2001,
Rna 7:471-82). Approximately 50% of successful transfection assays
resulted in the production of spliced mRNAs suggesting that a
subset of dendrites capable of splicing. As with any PCR-dependent
protocol, aberrant amplification of unspliced CDC pre-RNA or
spliced CDC RNA through mispriming could result in a truncated DNA
fragment that would mimic RNA splicing. If these data were the
result of such mispriming (internal priming of the CDC cDNA at
regions of homology in other CDC cDNAs), then we would expect that
a majority of transcripts contain three or more bases of similarity
in the donor and acceptor sites at the splice junction (the number
of 3'-end primer matched bases to prime PCR) (Wu et al., 1991, DNA
Cell Biol. 10:233-8; Liang et al., 1995, Curr. Opin. Immunol.
7:274-80). In contrast, 2/3 of the spliced sequences have two or
less bases overlap, suggesting that the PCR reaction was of high
fidelity. Furthermore, controls in all experiments showed that PCR
amplification of the CDC pre-RNA does not give rise to amplicons
distinct from the CDC pre-RNA template.
[0103] To show that this splicing event wasn't unique to the CDC
RNA, experiments were repeated using a second, independent
construct retaining the SV40 small t-antigen intron. The pGL-2
splicing construct (Promega, Carlsbad, Calif.) contains the
luciferase coding region fused to the SV40 small t-antigen intron
upstream of the luciferase polyadenylation site. This construct has
been used extensively in splicing experiments as a control where it
is efficiently spliced in mammalian cells. This luciferase-SV40
sequence was PCR amplified from the pGL-2 plasmid with the
5'-luciferase directed primer containing a T7 RNA polymerase
promoter site so that sense RNA can be made from this construct.
Following transfection splice product sequences from the
luciferase-SV40 chimera were analyzed as described for the CDC
pre-RNA splicing experiments using specific primers directed
against bases 1680-1700, 1700-1720,2580-2600, and 2680-2700. Five
unique spliced sequences were detected (FIG. 1). As observed with
CDC pre-RNA, both conventional and cryptic splice donor/acceptor
sites are observed. Cryptic splicing sites with the SV40 small
t-antigen intron 3' to some genes has been previously observed
(Evans et al., 1989, Gene 84:135-42; Huang et al., 1990, Mol. Cell
Biol. 10:1805-10). The characterized sequences are spliced forms
some of which show only two bases of `overlap` as per the pre-CDC
splicing discussion above. These data confirm and extend the data
from the CDC RNA transfection studies, and further show the RNA
splicing capacity of dendrites.
[0104] Amplification of spliced CDC pre-RNA from isolated, Sm
antigen-positive dendrites. To conclusively show that a dendrite
containing an identified splicing factor can also splice pre-RNA,
intact neurons were transfected with CDC pre-RNA, fixed at a later
time point with 4% paraformaldehyde, and the
dendritically-localized CDC RNA was copied into cDNA using an SP6
primer in situ (FIG. 5A). These dendrites were then
immunohistochemically stained for Sm proteins, and a single process
containing granule-like structures were harvested. The punctate
staining seen in FIG. 5 is qualitatively similar to that seen
previously (FIG. 2E). The in situ transcribed cDNA was isolated and
used as a PCR template for amplification of spliced CDC RNA. The
amplicons of spliced CDC RNA migrates faster than non-spliced CDC
pre-RNA during agarose gel electrophores (FIG. 5B) and shows splice
donor/acceptor sequence similarity to previous experiments (FIGS. 1
and 5). These data show that Sm antigen-positive processes are
capable of supporting dendritic pre-RNA splicing. In experiments
where Sm antigen was absent from dendrites, PCR amplification of
CDC pre-RNA transfected, Sm-negative, processes yielded no spliced
RNAs, suggesting that the inclusion of Sm antigen in dendrites is
correlated with the functional splicing activity in dendrites.
Experimental Example 3
Synaptoneurosomes can Splice CDC pre-RNA.
[0105] Synaptoneurosomes have been used to demonstrate protein
synthesis in dendritically enriched regions of neurons (Weiler et
al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:7168-71; Rao et al.,
1993, J. Neurochem. 61:835-44; Bagni et al., 2000, J. Neurosci.
20:RC76). This subcellular fraction of tissue homogenates contains
liposomes of pre- and post-synaptic entities. Visually, these
fractions are absent nuclei (Weiler et al., 1993, Proc. Natl. Acad.
Sci. U.S.A. 90:7168-71; Rao et al., 1993, J. Neurochem. 61:835-44;
Bagni et al., 2000, J. Neurosci. 20:RC76). Using established
protocols, we isolated fresh synaptoneurosomes from rat brain and
tested them for the presence of splicing proteins using western
blotting. As seen in FIG. 6, U2AF65, pan-SR, SC-35, SF2 and Sm
antigens were all present in the whole brain (WB) extract as well
as in the synaptoneurosome (SN) preparation. These results show
that these same pre-spliceosome proteins, localized in cultured
primary dendrites, are present in the WB and, more importantly, the
SN fraction at differing abundances. In this respect, the western
analysis data is consistent with the immunofluorescence data of
FIG. 2. While SN preparations are never purely neuronalin origin,
it is unlikely that the observed differing intensities of the
protein bands observed with chemiluminscence is function of nuclear
contamination as these preparations contain virtually no nuclei and
less than 5% mitochondria (Booth et al., 1978, Biochem. J.
176:365-70). Tubulin was used as a loading control to show that the
same amount of protein was loaded in the different lanes.
[0106] Given the presence of these proteins in SNs, the SN extracts
were tested for the ability to splice the CDC pre-RNA. Freshly
prepared SNs were supplemented with 1.5 mM ATP and incubated with
CDC pre-RNA for 30 minutes at 37.degree. C. After incubation, RNA
was isolated from the SNs and the spliced CDC RNA was PCR
amplified, cloned and sequenced. Ten spliced sequences generated
from three separate experiments are shown in FIG. 1. Consistent
with the ATP-dependent nature of the spliceosome and the relative
absence of mitochondria in these fractions, no splice forms were
generated when ATP was omitted from the SN preparation. A similar
effect was observed when SNs were subjected to a single freeze/thaw
cycle prior to the splicing assay.
Experimental Example 4
Spliced CDC RNA can be Translated in Isolated Dendrites.
[0107] As described previously, the isolated dendrite assay
involves mechanical severing of the dendrites from the cell soma,
removal of the soma, and transfection of the isolated dendrites.
FIGS. 7A, 7D and 7G show photomicrographs of primary rat
hippocampal neurons with the cell somas intact (white arrows).
FIGS. 7B, 7E and 7H show these same microscopic fields after
removal of the soma, leaving the dendrites (black arrows). Since
the CDC pre-RNA contains an in-frame FLAG epitope-tag in the second
exon this epitope should be immunohistochemically detectable if the
dendritically spliced CDC pre-RNA can be translated. Unspliced CDC
pre-RNA will not produce a translated FLAG epitope. These same
dendrites are stained for FLAG expression after mock transfection
(Panel C) and transfection with mature CDC RNA (Panel F) or
unspliced CDC pre-RNA (Panel I). These data show the similarity in
FLAG sequence expression levels in dendrites when transfected with
mature CDC RNA and CDC pre-RNA.
[0108] Dendritic transfection of CDC pre-RNA can result in the
isolation of alternatively spliced CDCRNAs that contain two
adjacent open reading frames (ORFs), with the 3' ORF containing the
FLAG epitope (FIG. 1). Therefore, as a result of splicing,
bicistronic mRNAs were created from the CDC pre-RNA. To assess
whether the FLAG-tags on downstream ORFs are translatable, the 236
bp (splice variant 1), 181 bp (splice variant 6), and 146 bp
(splice variant 45) cDNAs were selected and transcribed into RNA
that was then transfected into the isolated dendrites, followed by
immunostaining with the anti-FLAG antibody. The sequence of the 236
bp, 181 bp, and 146 bp cDNAs suggests that if the consensus splice
site is not utilized, the sequence extension of the 3'-end of the
first exon will give rise to an in-frame termination codon
producing a small translational unit. This 5' translational unit
does not contain the FLAG-tag. The 236 and 181 bp clones both
contain a second open reading frame that, if translated, would
contain the FLAG-tag. The 146 base sequence is spliced so that
there is no in-frame initiator methionine that could prime the
expression of the FLAG-tag. FIG. 8 shows that upon in vivo
translation, both the 236 bp and 181 bp alternatively spliced CDC
RNAs gave rise to FLAG antigenicity (FIGS. 8A-8B and 8C-8D,
respectively). Transfection of the 146 bp RNA did not produce FLAG
containing protein (FIGS. 8E and 8F). These data indicate that 1)
the in-frame methionine for the predicted second open-reading frame
produced by nontraditional splicing of the CDC pre-RNA can be
recognized by the dendritic protein synthesis machinery and
utilized as an initiator methionine to produce protein from the
second open-reading frame and 2) these transfected spliced RNAs can
be further spliced in the dendrite to yield a mature CDC RNA. The
translation of the second open-reading frame of a bicistronic mRNA
could occur by read-through of the second reading frame as the
ribosomes that translated the first open-reading frame move along
the RNA after termination of translation of the first open-reading
frame (Kozaket et al., 1998, Nucleic Acids Res. 26:4853-9).
Alternatively, translation could occur through utilization of an
internal ribosome entry site (IRES) that may be present in the
inter-cistronic region in a cap-independent process (Macejak et
al., 1991, Nature 353:90-4 [54]). M-fold analysis of the spliced
CDC RNAs reveals stem-loops and complex secondary structures that
have been implicated as potential IRES sites (Martinez-Salas et
al., 2002, Biochimie 84:755-63). Regardless of translational
mechanism, these data show that dendritically spliced RNAs can be
translated in the local dendritic environment.
Experimental Example 5
Control Data for GFP Fusion Constructs
[0109] Control experiments were conducted using the Olympus
Fluoview FV100 spectrometer to illustrate the specificity of the
GFP fusion constructs used in FIG. 4. As noted elsewhere herein,
the intensity of the laser was increased to facilitate imaging of
dendritic fluorescence. The fluorescence of the nuclei in these
experiments was saturated. In FIG. 10A, a representative image of
the "speckled" nuclear pattern of expression is shown using the
UAP56-GFP construct. Similar patterns of signal were observed for
all GFP fusion proteins exemplified herein. The images set forth in
FIG. 10 have been optimized for nuclear images so the intensity of
the laser is very low with no increase in gain. A ghosting of GFP
expression is visible in the perinuclear region and if the
intensity of the laser was increased dendritic staining would be
visible as well. In FIGS. 10B-10E are illustrated the two types of
non-neuronal staining that observed with GFP fusion constructs set
forth in detail elsewhere herein. In the first example, here
exemplified by SF1/mBBP-GFP (FIG. 10B) and its corresponding MAP2
immunofluorescence (FIG. 10C), expression is strongly localized to
the nucleus with very low levels of cytoplasmic signal. This type
of staining pattern was observed for UAP56-GFP and Y14-GFP as well.
A second non-neuronal RBP-GFP phenotype is visualized in FIG. 10D
with the U2AF65-GFP construct, in which can be observed a
well-defined expression in the nucleus with low, but significant
fluorescence visible throughout the glial cytoplasm. A similar
staining pattern is observed for Magoh-GFP in the glial cell of
these cultures (see also FIG. 4A). In FIG. 10E, a transfected
neuron (note the MAP2 fluorescence) is also visible in the lower
left quadrant of the image. The intensity of staining is apparent
in the neuron versus the glia. This difference in RBP-GFP
expression was typical when comparing basal levels of expression in
neurons and glia. Finally, the expression of the plain pEGFP-N1
construct is illustrated when expressed in glia (FIG. 10F) or
neurons (FIG. 10G). There is an obvious difference in the patterns
for non-neuronal cells when comparing FIG. 10F to FIGS. 10B and
10D. Similary, when GFP expression is neurons is not fused to a RBP
as described in the present disclosure, there are not distinct
patterns of expression. GFP is seen diffusely spread over the
nucleus and cytoplasm. This is in contrast to the patterns of
expression seen in FIGS. 2 and 4 illustrating the localization of
the native protein or the expression of a GFP fusion protein.
Finally, in FIGS. 10E-10H aer shown control experiments with
antigens that should retain a predominantly nuclear localization.
An anti-histone 3 antibody (E) and a histone 2B-YFP fusion
construct (G) were used to show the respective neuronal
localization. Corresponding phase-contrast photos (FIGS. 10F and
10H) illustrate the neuronal morphology. These data show that the
use of overexpressing GFP or YFP constructs do not, in all
circumstances, lead to localization in the dendritic fields. The
arrows in FIGS. 10E and 10F show where the dendrites are localized
in the images, while the arrows in FIG. 10G and 10H show the
dendrites in this image.
Experimental Example 6
Isolated Dendrite Viability Evaluation using Mitochondrial Function
Measurement
[0110] Dendrites were isolated from cortical cell cultures and at
various times after severing, JC-1 dye (Molecular Probes) was added
at a concentration of 1 .mu.g dye/ml media. The dye was incubated
at 37.degree. C. with the cultures for 10 min followed by imaging
with fluorescence microscopy. Functional mitochondria take up and
concentrate the green fluorescent monomer. Once the concentration
reached a threshold, the dye began to polymerize, forming red
fluorescent aggregates visualized as red puncta. A comparison of
the zero time point and the 5 hour time point show similar levels
of red fluorescence (FIG. 11).
[0111] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety.
[0112] While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and equivalent variations.
Sequence CWU 1
1
294 1 39 DNA Artificial reIF4A3 sense primer; chemically
synthesized 1 aatgaattcg ccaccatggc ggctaacgcc acgatggcg 39 2 36
DNA Artificial reIF4A3 antisense primer; chemically synthesized 2
atttggatcc cgaattaggt cagccacatt catggg 36 3 39 DNA Artificial
rMagoh sense primer; chemically synthesized 3 aataagcttg ccaccatgga
gagtgacttt tacctgcgt 39 4 36 DNA Artificial rMagoh antisense
primer; chemically synthesized 4 attgaccggt gggattggtt taatcttgaa
gtgtaa 36 5 39 DNA Artificial rRNPS1 sense primer; chemically
synthesized 5 aataagcttg ccaccatgga tttatcagga gtgaaaaag 39 6 33
DNA Artificial rRNPS1 antisense primer; chemically synthesized 6
attgaccggt gggagcagcc gtgaaccaac agt 33 7 39 DNA Artificial
rSF1/mBBP sense primer; chemically synthesized 7 aatgctagcg
ccaccatggc gaccggagcg aacgccacg 39 8 33 DNA Artificial rSF1/mBBP
antisense primer; chemically synthesized 8 atttggatcc caatgggcgc
ggaaagtcct cac 33 9 39 DNA Artificial rU2AF65 sense primer;
chemically synthesized 9 aataagcttg ccaccatgga cttcttcaac gcccagatg
39 10 33 DNA Artificial rU2AF65 antisense primer; chemically
synthesized 10 atttggatcc cagaagtccc gacggtggta cga 33 11 39 DNA
Artificial rUAP56 sense primer; chemically synthesized 11
aataagcttg ccaccatggc agagaacgat gtggacaat 39 12 33 DNA Artificial
rUAP56 antisense primer; chemically synthesized 12 atttggatcc
cgtgtctgtt caatgtagga gga 33 13 39 DNA Artificial rY14 sense
primer; chemically synthesized 13 aataagcttg ccaccatggc ggacgtgctg
gatcttcac 39 14 36 DNA Artificial rY14 antisense primer; chemically
synthesized 14 atttggatcc cgacggcgtc tccggtctgg actcct 36 15 19 DNA
Artificial Sp6 primer; chemically synthesized 15 atttaggtga
cactataga 19 16 19 DNA Artificial FLAG primer; chemically
synthesized 16 tttatcgtca tcgtctttg 19 17 19 DNA Artificial 5A
primer; chemically synthesized 17 ccaatcgata tacttagcc 19 18 21 DNA
Artificial 5B primer; chemically synthesized 18 gccagtgcca
agcttgctga c 21 19 15 DNA Artificial sequence fragment of CDC RNA
splice product 19 ttaagtgttg tacag 15 20 15 DNA Artificial sequence
fragment of CDC RNA splice product 20 atacgagagc tctag 15 21 15 DNA
Artificial sequence fragment of CDC RNA splice product 21
ctttgagaca ctaac 15 22 15 DNA Artificial sequence fragment of CDC
RNA splice product 22 gtgtccccag ggagc 15 23 15 DNA Artificial
sequence fragment of CDC RNA splice product 23 tctcattatt taatg 15
24 15 DNA Artificial sequence fragment of CDC RNA splice product 24
tggttggagg acaca 15 25 15 DNA Artificial sequence fragment of CDC
RNA splice product 25 catggagaag gctct 15 26 15 DNA Artificial
sequence fragment of CDC RNA splice product 26 gacccctgag ttgct 15
27 15 DNA Artificial sequence fragment of CDC RNA splice product 27
ctgtgctcca ggttg 15 28 15 DNA Artificial sequence fragment of CDC
RNA splice product 28 ccactggagt gattt 15 29 15 DNA Artificial
sequence fragment of CDC RNA splice product 29 aggagaacat ggaga 15
30 15 DNA Artificial sequence fragment of CDC RNA splice product 30
aggctctgac ccctg 15 31 15 DNA Artificial sequence fragment of CDC
RNA splice product 31 tgccactgga gtgat 15 32 15 DNA Artificial
sequence fragment of CDC RNA splice product 32 ttctaccctc caggt 15
33 15 DNA Artificial sequence fragment of CDC RNA splice product 33
catggagaag gctct 15 34 15 DNA Artificial sequence fragment of CDC
RNA splice product 34 gacccctgag ttgct 15 35 15 DNA Artificial
sequence fragment of CDC RNA splice product 35 aaggtttcag cttct 15
36 15 DNA Artificial sequence fragment of CDC RNA splice product 36
cattatttaa tgtgg 15 37 15 DNA Artificial sequence fragment of CDC
RNA splice product 37 tccttttgca ggtca 15 38 15 DNA Artificial
sequence fragment of CDC RNA splice product 38 acaaggagaa catgg 15
39 15 DNA Artificial sequence fragment of CDC RNA splice product 39
cgagagctct agtct 15 40 15 DNA Artificial sequence fragment of CDC
RNA splice product 40 ggtcctaaca tgaag 15 41 15 DNA Artificial
sequence fragment of CDC RNA splice product 41 tgcaggtcaa caagg 15
42 15 DNA Artificial sequence fragment of CDC RNA splice product 42
agaacatgga gaagg 15 43 15 DNA Artificial sequence fragment of CDC
RNA splice product 43 cattatttaa tgtgg 15 44 15 DNA Artificial
sequence fragment of CDC RNA splice product 44 ttggaggaca cattt 15
45 15 DNA Artificial sequence fragment of CDC RNA splice product 45
ctgcctctcc tcttt 15 46 15 DNA Artificial sequence fragment of CDC
RNA splice product 46 gcaggtcaac aagga 15 47 15 DNA Artificial
sequence fragment of CDC RNA splice product 47 ttgcgtgttg tacag 15
48 15 DNA Artificial sequence fragment of CDC RNA splice product 48
atacgagagc tctag 15 49 15 DNA Artificial sequence fragment of CDC
RNA splice product 49 ctttgagaca ctaac 15 50 15 DNA Artificial
sequence fragment of CDC RNA splice product 50 gtgtccccag ggagc 15
51 15 DNA Artificial sequence fragment of CDC RNA splice product 51
ctggagtgat ttcta 15 52 15 DNA Artificial sequence fragment of CDC
RNA splice product 52 ccctccaggt aaggt 15 53 15 DNA Artificial
sequence fragment of CDC RNA splice product 53 ctctctctct gcctc 15
54 15 DNA Artificial sequence fragment of CDC RNA splice product 54
tcctctttgc aggtc 15 55 15 DNA Artificial sequence fragment of CDC
RNA splice product 55 tttctaccct ccagg 15 56 15 DNA Artificial
sequence fragment of CDC RNA splice product 56 taaggtttca gcttc 15
57 15 DNA Artificial sequence fragment of CDC RNA splice product 57
ctctttgcag gtcaa 15 58 15 DNA Artificial sequence fragment of CDC
RNA splice product 58 caaggagaac atgga 15 59 15 DNA Artificial
sequence fragment of CDC RNA splice product 59 tctggtccta acatg 15
60 15 DNA Artificial sequence fragment of CDC RNA splice product 60
ctgacccctc actcc 15 61 15 DNA Artificial sequence fragment of CDC
RNA splice product 61 aacatggaga aggct 15 62 15 DNA Artificial
sequence fragment of CDC RNA splice product 62 ctgacccctg agttg 15
63 15 DNA Artificial sequence fragment of CDC RNA splice product 63
ccactggagt gattt 15 64 15 DNA Artificial sequence fragment of CDC
RNA splice product 64 ctaccctcca ggtaa 15 65 15 DNA Artificial
sequence fragment of CDC RNA splice product 65 ctttgcaggt caaca 15
66 15 DNA Artificial sequence fragment of CDC RNA splice product 66
aggagaacat ggaga 15 67 15 DNA Artificial sequence fragment of CDC
RNA splice product 67 acctgctgac tgctg 15 68 15 DNA Artificial
sequence fragment of CDC RNA splice product 68 tgctccaggt tgcca 15
69 15 DNA Artificial sequence fragment of CDC RNA splice product 69
caaggagaac atgga 15 70 15 DNA Artificial sequence fragment of CDC
RNA splice product 70 gaaggctctg acccc 15 71 15 DNA Artificial
sequence fragment of CDC RNA splice product 71 atgtggttgg aggac 15
72 15 DNA Artificial sequence fragment of CDC RNA splice product 72
cattttaagt gttgt 15 73 15 DNA Artificial sequence fragment of CDC
RNA splice product 73 ctcctctttg caggt 15 74 15 DNA Artificial
sequence fragment of CDC RNA splice product 74 caacaaggag aacat 15
75 15 DNA Artificial sequence fragment of CDC RNA splice product 75
aggttgccac tggag 15 76 15 DNA Artificial sequence fragment of CDC
RNA splice product 76 tgatttctac cctcc 15 77 15 DNA Artificial
sequence fragment of CDC RNA splice product 77 gcctctcctc tttgc 15
78 15 DNA Artificial sequence fragment of CDC RNA splice product 78
aggtcaacaa ggaga 15 79 15 DNA Artificial sequence fragment of CDC
RNA splice product 79 gtttcagctt ctcat 15 80 15 DNA Artificial
sequence fragment of CDC RNA splice product 80 tatttaatgt ggttg 15
81 15 DNA Artificial sequence fragment of CDC RNA splice product 81
atggagaagg ctctg 15 82 15 DNA Artificial sequence fragment of CDC
RNA splice product 82 acccctgagt tgctg 15 83 15 DNA Artificial
sequence fragment of CDC RNA splice product 83 ctctagtctg gtcct 15
84 15 DNA Artificial sequence fragment of CDC RNA splice product 84
aacatgaaga cttgc 15 85 15 DNA Artificial sequence fragment of CDC
RNA splice product 85 aacaaggaga acatg 15 86 15 DNA Artificial
sequence fragment of CDC RNA splice product 86 gagaaggctc tgacc 15
87 15 DNA Artificial sequence fragment of CDC RNA splice product 87
ctgtgctcca ggttg 15 88 15 DNA Artificial sequence fragment of CDC
RNA splice product 88 ccactggagt gattt 15 89 15 DNA Artificial
sequence fragment of CDC RNA splice product 89 caaggagaac atgga 15
90 15 DNA Artificial sequence fragment of CDC RNA splice product 90
gaaggctctg acccc 15 91 15 DNA Artificial sequence fragment of CDC
RNA splice product 91 ctcattattt aatgt 15 92 15 DNA Artificial
sequence fragment of CDC RNA splice product 92 ggttggagga cacat 15
93 15 DNA Artificial sequence fragment of CDC RNA splice product 93
agaaggctct gaccc 15 94 15 DNA Artificial sequence fragment of CDC
RNA splice product 94 ctgagttgct gtcta 15 95 15 DNA Artificial
sequence fragment of CDC RNA splice product 95 cttctcatta tttaa 15
96 15 DNA Artificial sequence fragment of CDC RNA splice product 96
tgtggttgga ggaca 15 97 15 DNA Artificial sequence fragment of CDC
RNA splice product 97 ggagaaggct ctgac 15 98 15 DNA Artificial
sequence fragment of CDC RNA splice product 98 ccctgagttg ctgtc 15
99 15 DNA Artificial sequence fragment of CDC RNA splice product 99
agtaaggttt cagct 15 100 16 DNA Artificial sequence fragment of CDC
RNA splice product 100 tctcattatt taatgt 16 101 15 DNA Artificial
sequence fragment of CDC RNA splice product 101 gttgtgtcta ctgat 15
102 15 DNA Artificial sequence fragment of CDC RNA splice product
102 cggggtagac tacaa 15 103 15 DNA Artificial sequence fragment of
CDC RNA splice product 103 gactgctgtg ctcca 15 104 15 DNA
Artificial sequence fragment of CDC RNA splice product 104
ggttgccact ggagt 15 105 15 DNA Artificial sequence fragment of CDC
RNA splice product 105 ctctttgcag gtcaa 15 106 15 DNA Artificial
sequence fragment of CDC RNA splice product 106 caaggagaac atgga 15
107 15 DNA Artificial sequence fragment of CDC RNA splice product
107 tggagtgatt tctac 15 108 15 DNA Artificial sequence fragment of
CDC RNA splice product 108 cctccaggta aggtt 15 109 15 DNA
Artificial sequence fragment of CDC RNA splice product 109
gcaaatgata acctc 15 110 15 DNA Artificial sequence fragment of CDC
RNA splice product 110 tctctctgcc tctcc 15 111 15 DNA Artificial
sequence fragment of CDC RNA splice product 111 tagtctggtc ctaac 15
112 15 DNA Artificial sequence fragment of CDC RNA splice product
112 atgaagactt gctca 15 113 15 DNA Artificial sequence fragment of
CDC RNA splice product 113 gagaaggctc tgacc 15 114 15 DNA
Artificial sequence fragment of CDC RNA splice product 114
cctgagttgc tgtct 15 115 15 DNA Artificial sequence fragment of CDC
RNA splice product 115 catgaagact tgctc 15 116 15 DNA Artificial
sequence fragment of CDC RNA splice product 116 actcctactg cttgt 15
117 15 DNA Artificial sequence fragment of CDC RNA splice product
117 ggagaaggct ctgac 15 118 15 DNA Artificial sequence fragment of
CDC RNA splice product 118 ccctgagttg ctgtc 15 119 15 DNA
Artificial sequence fragment of CDC RNA splice product 119
ggacacattt taagt 15 120 15 DNA Artificial sequence fragment of CDC
RNA splice product 120 gttgtacaga tacga 15 121 15 DNA Artificial
sequence fragment of CDC RNA splice product 121 tctctctctg cctct 15
122 15 DNA Artificial sequence fragment of CDC RNA splice product
122 cctctttgca ggtca 15 123 15 DNA Artificial sequence fragment of
CDC RNA splice product 123 gagtgatttc taccc 15 124 15 DNA
Artificial sequence fragment of CDC RNA splice product 124
tccaggtaag gtttc 15 125 15 DNA Artificial sequence fragment of CDC
RNA splice product 125 ggagaaggct ctgac 15 126 15 DNA Artificial
sequence fragment of CDC RNA splice product 126 ccctgagttg
ctgtc
15 127 15 DNA Artificial sequence fragment of CDC RNA splice
product 127 aggacacatt ttaag 15 128 15 DNA Artificial sequence
fragment of CDC RNA splice product 128 tgttgtacag atacg 15 129 15
DNA Artificial sequence fragment of CDC RNA splice product 129
aggctctgac ccctg 15 130 15 DNA Artificial sequence fragment of CDC
RNA splice product 130 agttgctgtc tactg 15 131 15 DNA Artificial
sequence fragment of CDC RNA splice product 131 tgctgtgctc caggt 15
132 15 DNA Artificial sequence fragment of CDC RNA splice product
132 tgccactgga gtgat 15 133 15 DNA Artificial sequence fragment of
CDC RNA splice product 133 gacccctgag ttgct 15 134 15 DNA
Artificial sequence fragment of CDC RNA splice product 134
gtctactgat cgggt 15 135 15 DNA Artificial sequence fragment of CDC
RNA splice product 135 gagtgatttc taccc 15 136 15 DNA Artificial
sequence fragment of CDC RNA splice product 136 ccctgtaagg tttca 15
137 15 DNA Artificial sequence fragment of CDC RNA splice product
137 agaaggctct gaccc 15 138 15 DNA Artificial sequence fragment of
CDC RNA splice product 138 ccctgagttg ctgtc 15 139 15 DNA
Artificial sequence fragment of CDC RNA splice product 139
gttgtacaga tacga 15 140 15 DNA Artificial sequence fragment of CDC
RNA splice product 140 gctctagtct ggtcc 15 141 15 DNA Artificial
sequence fragment of CDC RNA splice product 141 aggagaacat ggaga 15
142 15 DNA Artificial sequence fragment of CDC RNA splice product
142 aggctctgac ccctg 15 143 15 DNA Artificial sequence fragment of
CDC RNA splice product 143 agtaaggttt cagct 15 144 15 DNA
Artificial sequence fragment of CDC RNA splice product 144
tctcattatt taatg 15 145 15 DNA Artificial sequence fragment of CDC
RNA splice product 145 caaatgataa cctct 15 146 15 DNA Artificial
sequence fragment of CDC RNA splice product 146 ctctctgcct ctcct 15
147 15 DNA Artificial sequence fragment of CDC RNA splice product
147 aggtaaggtt tcagc 15 148 15 DNA Artificial sequence fragment of
CDC RNA splice product 148 ctctcctctt tgcag 15 149 15 DNA
Artificial sequence fragment of CDC RNA splice product 149
aacctctctc tctgc 15 150 15 DNA Artificial sequence fragment of CDC
RNA splice product 150 ctctcctctt tgcag 15 151 15 DNA Artificial
sequence fragment of CDC RNA splice product 151 tgactgctgt gctcc 15
152 15 DNA Artificial sequence fragment of CDC RNA splice product
152 caggttgcca ctgga 15 153 15 DNA Artificial sequence fragment of
CDC RNA splice product 153 gagaaggctc tgacc 15 154 15 DNA
Artificial sequence fragment of CDC RNA splice product 154
cctgagttgc tgtct 15 155 15 DNA Artificial sequence fragment of CDC
RNA splice product 155 tttctaccct ccagg 15 156 15 DNA Artificial
sequence fragment of CDC RNA splice product 156 taaggtttca gcttc 15
157 15 DNA Artificial sequence fragment of CDC RNA splice product
157 agaacatgga gaagg 15 158 15 DNA Artificial sequence fragment of
CDC RNA splice product 158 ctctgacccc tgagt 15 159 15 DNA
Artificial sequence fragment of CDC RNA splice product 159
tgctgtgctc caggt 15 160 15 DNA Artificial sequence fragment of CDC
RNA splice product 160 gccactggag tgatt 15 161 15 DNA Artificial
sequence fragment of CDC RNA splice product 161 gtctactgat cgggt 15
162 15 DNA Artificial sequence fragment of CDC RNA splice product
162 agactacaaa gacga 15 163 15 DNA Artificial sequence fragment of
CDC RNA splice product 163 gatttctacc cccca 15 164 15 DNA
Artificial sequence fragment of CDC RNA splice product 164
tgtaaggttt cagct 15 165 15 DNA Artificial sequence fragment of CDC
RNA splice product 165 ccttgatgaa gtcca 15 166 15 DNA Artificial
sequence fragment of CDC RNA splice product 166 ttctttgaga cacta 15
167 15 DNA Artificial sequence fragment of CDC RNA splice product
167 ggtttcagct tctca 15 168 15 DNA Artificial sequence fragment of
CDC RNA splice product 168 ttatttaatg tggtt 15 169 15 DNA
Artificial sequence fragment of CDC RNA splice product 169
cctctttgca ggtca 15 170 15 DNA Artificial sequence fragment of CDC
RNA splice product 170 acaaggagaa catgg 15 171 15 DNA Artificial
sequence fragment of CDC RNA splice product 171 ctctagtctg gtcct 15
172 15 DNA Artificial sequence fragment of CDC RNA splice product
172 aacatgaaga cttgc 15 173 15 DNA Artificial sequence fragment of
CDC RNA splice product 173 aaggctctga cccct 15 174 15 DNA
Artificial sequence fragment of CDC RNA splice product 174
gagttgctgt ctact 15 175 15 DNA Artificial sequence fragment of CDC
RNA splice product 175 gctgactgct gtgct 15 176 15 DNA Artificial
sequence fragment of CDC RNA splice product 176 ccaggttgcc actgg 15
177 15 DNA Artificial sequence fragment of CDC RNA splice product
177 gacccctgag ttgct 15 178 15 DNA Artificial sequence fragment of
CDC RNA splice product 178 gtctactgat cgggt 15 179 15 DNA
Artificial sequence fragment of CDC RNA splice product 179
gtgatttcta ccctc 15 180 15 DNA Artificial sequence fragment of CDC
RNA splice product 180 caggtaaggt ttcag 15 181 15 DNA Artificial
sequence fragment of CDC RNA splice product 181 ctctctctct gcctc 15
182 15 DNA Artificial sequence fragment of CDC RNA splice product
182 tcctctttgc aggtc 15 183 15 DNA Artificial sequence fragment of
CDC RNA splice product 183 tgatttctac cctcc 15 184 15 DNA
Artificial sequence fragment of CDC RNA splice product 184
tgtaaggttt cagct 15 185 15 DNA Artificial sequence fragment of CDC
RNA splice product 185 tctctctgcc tctcc 15 186 15 DNA Artificial
sequence fragment of CDC RNA splice product 186 tctttgcagg tcaac 15
187 15 DNA Artificial sequence fragment of CDC RNA splice product
187 tatttaatgt ggttg 15 188 15 DNA Artificial sequence fragment of
CDC RNA splice product 188 gaggacacat tttaa 15 189 15 DNA
Artificial sequence fragment of CDC RNA splice product 189
ctgacccctg agttg 15 190 15 DNA Artificial sequence fragment of CDC
RNA splice product 190 ctgtctactg atcgg 15 191 15 DNA Artificial
sequence fragment of CDC RNA splice product 191 gtgatttcta ccctc 15
192 15 DNA Artificial sequence fragment of CDC RNA splice product
192 caggtaaggt ttcag 15 193 15 DNA Artificial sequence fragment of
CDC RNA splice product 193 tctctgcctc tcctc 15 194 15 DNA
Artificial sequence fragment of CDC RNA splice product 194
tttgcaggtc aacaa 15 195 15 DNA Artificial sequence fragment of CDC
RNA splice product 195 ctggtcctaa catga 15 196 15 DNA Artificial
sequence fragment of CDC RNA splice product 196 agacttgctc actcc 15
197 15 DNA Artificial sequence fragment of CDC RNA splice product
197 gggagcagca aatga 15 198 15 DNA Artificial sequence fragment of
CDC RNA splice product 198 taacctctct ctctg 15 199 15 DNA
Artificial sequence fragment of CDC RNA splice product 199
accctccagg taagg 15 200 15 DNA Artificial sequence fragment of CDC
RNA splice product 200 tttcagcttc tcatt 15 201 15 DNA Artificial
sequence fragment of CDC RNA splice product 201 tgcaggtcaa caagg 15
202 15 DNA Artificial sequence fragment of CDC RNA splice product
202 agaacatgga gaagg 15 203 15 DNA Artificial sequence fragment of
CDC RNA splice product 203 tttaatgtga agact 15 204 15 DNA
Artificial sequence fragment of CDC RNA splice product 204
tgctcactcc tactg 15 205 15 DNA Artificial sequence fragment of CDC
RNA splice product 205 ctgatcgggt agact 15 206 15 DNA Artificial
sequence fragment of CDC RNA splice product 206 acaaagacga tgacg 15
207 15 DNA Artificial sequence fragment of CDC RNA splice product
207 tgctgtgctc caggt 15 208 15 DNA Artificial sequence fragment of
CDC RNA splice product 208 tgccactgga gtgat 15 209 15 DNA
Artificial sequence fragment of CDC RNA splice product 209
ctcctctttg caggt 15 210 15 DNA Artificial sequence fragment of CDC
RNA splice product 210 caacaaggag aacat 15 211 15 DNA Artificial
sequence fragment of CDC RNA splice product 211 tctggtccta acatg 15
212 15 DNA Artificial sequence fragment of CDC RNA splice product
212 aagacttgcg tgttg 15 213 15 DNA Artificial sequence fragment of
CDC RNA splice product 213 aacaaggaga acatg 15 214 15 DNA
Artificial sequence fragment of CDC RNA splice product 214
gagaaggctc tgacc 15 215 15 DNA Artificial sequence fragment of CDC
RNA splice product 215 gtgccaagct tgctg 15 216 15 DNA Artificial
sequence fragment of CDC RNA splice product 216 actgctgtgc tccag 15
217 15 DNA Artificial sequence fragment of CDC RNA splice product
217 acccctgagt tgctg 15 218 15 DNA Artificial sequence fragment of
CDC RNA splice product 218 tctactgatc gggta 15 219 15 DNA
Artificial sequence fragment of CDC RNA splice product 219
cttgctgact gctgt 15 220 15 DNA Artificial sequence fragment of CDC
RNA splice product 220 gctccaggtt gccac 15 221 15 DNA Artificial
sequence fragment of CDC RNA splice product 221 cccctgagtt gctgt 15
222 15 DNA Artificial sequence fragment of CDC RNA splice product
222 ctactgatcg ggtag 15 223 15 DNA Artificial sequence fragment of
CDC RNA splice product 223 ttgctactcc tactg 15 224 15 DNA
Artificial sequence fragment of CDC RNA splice product 224
cttgttatga cccca 15 225 15 DNA Artificial sequence fragment of CDC
RNA splice product 225 agttgctgtc tactg 15 226 15 DNA Artificial
sequence fragment of CDC RNA splice product 226 atcgggtaga ctaca 15
227 15 DNA Artificial sequence fragment of CDC RNA splice product
227 tctggtccta acatg 15 228 15 DNA Artificial sequence fragment of
CDC RNA splice product 228 aagacttgct cactc 15 229 15 DNA
Artificial sequence fragment of CDC RNA splice product 229
aacaaggaga acatg 15 230 15 DNA Artificial sequence fragment of CDC
RNA splice product 230 gagaaggctc tgacc 15 231 15 DNA Artificial
sequence fragment of CDC RNA splice product 231 tttaatgtga agact 15
232 15 DNA Artificial sequence fragment of CDC RNA splice product
232 tgctcactcc tactg 15 233 15 DNA Artificial sequence fragment of
CDC RNA splice product 233 ctgatcgggt agact 15 234 15 DNA
Artificial sequence fragment of CDC RNA splice product 234
acaaagacga tgacg 15 235 15 DNA Artificial sequence fragment of CDC
RNA splice product 235 aaagtccaaa ttgta 15 236 15 DNA Artificial
sequence fragment of CDC RNA splice product 236 ccaaattgta aaatg 15
237 15 DNA Artificial sequence fragment of CDC RNA splice product
237 tactgttttt tctta 15 238 15 DNA Artificial sequence fragment of
CDC RNA splice product 238 ctccacacag gcata 15 239 15 DNA
Artificial sequence fragment of CDC RNA splice product 239
aagtccaaat tgtaa 15 240 15 DNA Artificial sequence fragment of CDC
RNA splice product 240 aatgtaactg tattc 15 241 15 DNA Artificial
sequence fragment of CDC RNA splice product 241 cagttataat cataa 15
242 15 DNA Artificial sequence fragment of CDC RNA splice product
242 catactgttt tttct 15 243 15 DNA Artificial sequence fragment of
CDC RNA splice product 243 ttacgtcgcc agtca 15 244 15 DNA
Artificial sequence fragment of CDC RNA splice product 244
agtaacaacc gcgaa 15 245 15 DNA Artificial sequence fragment of CDC
RNA splice product 245 taacagttat aatca 15 246 15 DNA Artificial
sequence fragment of CDC RNA splice product 246 taacatactg ttttt 15
247 15 DNA Artificial sequence fragment of CDC RNA splice product
247 cttaccggaa aactc 15 248 15 DNA Artificial sequence fragment of
CDC RNA splice product 248 gacgcaagaa aaatc 15 249 15 DNA
Artificial sequence fragment of CDC RNA splice product 249
tgttttttct tactc 15 250 15 DNA Artificial sequence fragment of CDC
RNA splice product 250 cacacaggca tagag 15 251 15 DNA Artificial
sequence fragment of CDC RNA splice product 251 gatgacggaa aaaga 15
252 15 DNA Artificial
sequence fragment of CDC RNA splice product 252 gatcgtggat tacgt 15
253 15 DNA Artificial sequence fragment of CDC RNA splice product
253 ctcctccaaa aaaga 15 254 15 DNA Artificial sequence fragment of
CDC RNA splice product 254 agagaaaggt agaag 15 255 15 DNA
Artificial sequence fragment of CDC RNA splice product 255
cttgctcact cctac 15 256 15 DNA Artificial sequence fragment of CDC
RNA splice product 256 tgcttgttat gaccc 15 257 15 DNA Artificial
sequence fragment of CDC RNA splice product 257 ctactgatcg ggtag 15
258 15 DNA Artificial sequence fragment of CDC RNA splice product
258 actacaaaga cgatg 15 259 15 DNA Artificial sequence fragment of
CDC RNA splice product 259 ttttaagtgt tgtac 15 260 15 DNA
Artificial sequence fragment of CDC RNA splice product 260
agatacgaga gctct 15 261 15 DNA Artificial sequence fragment of CDC
RNA splice product 261 tctctgcctc tcctc 15 262 15 DNA Artificial
sequence fragment of CDC RNA splice product 262 gataacctct ctctc 15
263 15 DNA Artificial sequence fragment of CDC RNA splice product
263 ttcagcttct catta 15 264 15 DNA Artificial sequence fragment of
CDC RNA splice product 264 tttaatgtgg ttgga 15 265 15 DNA
Artificial sequence fragment of CDC RNA splice product 265
tcctctttgc aggtc 15 266 15 DNA Artificial sequence fragment of CDC
RNA splice product 266 aacaaggaga acatg 15 267 15 DNA Artificial
sequence fragment of CDC RNA splice product 267 aggtttcagc ttctc 15
268 15 DNA Artificial sequence fragment of CDC RNA splice product
268 attatttaat gtggt 15 269 15 DNA Artificial sequence fragment of
CDC RNA splice product 269 cctctctctc tgcct 15 270 15 DNA
Artificial sequence fragment of CDC RNA splice product 270
ctcctctttg caggt 15 271 15 DNA Artificial sequence fragment of CDC
RNA splice product 271 ctgactgctg tgctc 15 272 15 DNA Artificial
sequence fragment of CDC RNA splice product 272 caggttgcca ctgga 15
273 15 DNA Artificial sequence fragment of CDC RNA splice product
273 ccccaccaca ggcag 15 274 15 DNA Artificial sequence fragment of
CDC RNA splice product 274 ctcagataca cttgg 15 275 15 DNA
Artificial sequence fragment of CDC RNA splice product 275
cctccaggta aggtt 15 276 15 DNA Artificial sequence fragment of CDC
RNA splice product 276 tcagcttctc attat 15 277 15 DNA Artificial
sequence fragment of CDC RNA splice product 277 tgctgtctac tgatc 15
278 15 DNA Artificial sequence fragment of CDC RNA splice product
278 gggtagacta caaag 15 279 15 DNA Artificial sequence fragment of
CDC RNA splice product 279 aggtttcagc ttctc 15 280 15 DNA
Artificial sequence fragment of CDC RNA splice product 280
attatttaat gtggt 15 281 15 DNA Artificial sequence fragment of CDC
RNA splice product 281 aaatgataac ctctc 15 282 15 DNA Artificial
sequence fragment of CDC RNA splice product 282 tctctgcctc tcctc 15
283 15 DNA Artificial sequence fragment of CDC RNA splice product
283 gcttgttatg acccc 15 284 16 DNA Artificial sequence fragment of
CDC RNA splice product 284 accacaggca gctcag 16 285 15 DNA
Artificial sequence fragment of CDC RNA splice product 285
acatggagaa ggctc 15 286 15 DNA Artificial sequence fragment of CDC
RNA splice product 286 tgacccctga gttgc 15 287 15 DNA Artificial
sequence fragment of CDC RNA splice product 287 ttcagcttct catta 15
288 15 DNA Artificial sequence fragment of CDC RNA splice product
288 tttaatgtgg ttgga 15 289 15 DNA Artificial sequence fragment of
CDC RNA splice product 289 cattctttga gacac 15 290 15 DNA
Artificial sequence fragment of CDC RNA splice product 290
taacgtgtcc ccagg 15 291 15 DNA Artificial sequence fragment of CDC
RNA splice product 291 cttgctgact gctgt 15 292 15 DNA Artificial
sequence fragment of CDC RNA splice product 292 gctccaggtt gccac 15
293 15 DNA Artificial sequence fragment of CDC RNA splice product
293 ctctgacccc tgagt 15 294 29 DNA Artificial sequence fragment of
CDC RNA splice product misc_feature (17)..(17) n is a, c, g, or t
misc_feature (19)..(19) n is a, c, g, or t misc_feature (22)..(22)
n is a, c, g, or t misc_feature (24)..(24) n is a, c, g, or t
misc_feature (26)..(26) n is a, c, g, or t misc_feature (28)..(28)
n is a, c, g, or t 294 tgctgtctac tgatccntnd cntndnsns 29
* * * * *