ALTERNATIVE SPLICING

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ALTERNATIVE SPLICING
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ALTERNATIVE SPLICING

• Alternative splicing of mRNA precursors is a
  versatile mechanism of gene expression regulation
  that accounts for a considerable proportion of
  proteomic complexity in higher eukaryotes.
• Its modulation is achieved through the interplay
  of positive and negative regulatory signals
  present in the RNA, which are recognized by
  complexes composed of members of the hnRNP and SR
  protein families.

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It was recently predicted that around 60 of all
human genes undergo at least one process of alter
native splicing. The ever increasing known examp
les of alternative pre-mRNA processing are often
tissue type or developmental state specific,
indicating that complex regulation is involved in
the selection of SPLICE SITE pairs.
Lander, E. S., et al. 2001. Initial sequencing
and analysis of the human genome. Nature
409860921.
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Alternative splicing

• Number of Publications on Alternative Splicing in
  the PubMed data base
  
• 19,630

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Possible Splicing Patterns for DSCAM
38,016 Shades of DSCAM The DSCAM gene (top) is 61
.2 kb long and after transcription and splicing
produces a 7.8 kb, 24 exon mRNA (middle). Exons
4, 6, 9, and 17 are encoded as arrays of mutually
exclusive alternative exons. Each mRNA will
contain one of 12 possible alternatives for exon
4 (in red), one of 48 for exon 6 (blue), one of
33 for exon 9 (green) and one of 2 for exon 17
(yellow). In the final protein product (bottom),
exon 4 encodes the amino-terminal half of Ig
domain 2. Exon 6 encodes the same transmembrane
portion of Ig domain 3, exon 9 encodes all of Ig
domain 7, and 17 encodes the transmembrane
domain. If all possible combinations of single
exons 4, 6, 9, and 17 are used, the DSCAM gene
produces 38,016 different mRNAs and proteins.
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Positive Regulation
Small T
ASF/ SF2
UAG
Large T
Example SV40 large T antigen vs. small T
antigen. The normal ratio of expression of large
T to small T is 101. However, in 293-T cells,
the ratio is 11. The reason is that the SR prot
ein ASF/SF2 is expressed at 10 times the levels
in 293-T cells compared to HeLa cells. ASF/SF2
binds to the small T-antigen 5SS to select it.
This attracts the U1snRNP to the small T 5
splice site, which is a weaker site than the
large T 5 splice site. Positive regulation.
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Negative Regulation
Tra
male
yyyyy
UAG
yyyyy
U2AF
female
In Drosophila sexual development, the sex lethal
protein is one of earliest gene products in the
pathway. Sxl binds to the polypyrimidine tract i
n the male default pathway at 100x higher
affinity than to the female polypyrimidine tract.
So sxl out-competes U2AF. Consequently, U2AF bin
ds to the female site and you get the regulated
female mRNA. The male Tra protein is inactive be
cause of the UAG.
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Tra-2 autoregulates its own pre-mRNA splicing
UAG
NO splicing
Tra-2 is an SR splicing regulatory protein in
Drosophila. Tra-2 binds to the 5 splice site a
nd prevents the U1 snRNP from binding so that
there is no spliceosome assembly and No splicing.
The intron is retained. This results in a
truncated Tra-2 protein so functional Tra-2
protein levels decrease. This results in the mal
e default splicing of the intron and protein
levels go up. The cycle of regulation begins
again.
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Sex Lethal (sxl)- autoregulation
male
UAG
U2AF
female
Female splicing is regulated.
Sxl binds to the polypyrimide tract of the male
3 splice site of exon m so that this exon is
skipped because U2AF cannot bind.
Male default splicing. The exon is included and
the protein is inactive. Inactive sxl cant bind
the 3 splice site.
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Rat alpha-tropomyosin
Never
1
2
4
2
3
In muscle cells
In non muscle cells
1
3
4
The branch point of intron 2 is 177 nt upstream
of the 3 SS and is less than 40 nt from the 5
SS. This distance is too short for the formation
of the spliceosome because of steric
constraints. Therefore, exon 1 gets spliced to e
xon 3 then exon 4 or exon 1 gets spliced to exon
2 then exon 4.
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Double sex (dsx) Positive regulation by an Exonic
Enhancer Element
male
ESE
female
Alternative splicing and alternative poly(A)
sites. Female exon 3 contains a sequence element
termed an Exonic Splicing Enhancer (ESE).
It is a 13 nt purine-rich sequence that is
repeated 6 times. Two Drosophila SR proteins, tr
a and tra-2 bind to the female ESE and by
protein-protein interactions, attract U2AF to the
otherwise weak female exon 3 3SS.
SR proteins can function to assemble splicing
complexes at weak 5 or 3 SS by binding to ESE
and then attracting U1 snRNP or U2AF to foster
spliceosome assembly at the otherwise weak site.
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Different modes of alternative splicing and its
biological consequences.
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CONSEQUENCES OF REGULATED PRE-mRNA SPLICING IN
THE IMMUNE SYSTEMK. W. LynchNATURE REVIEWS
IMMUNOLOGYVOLUME 4 DECEMBER 2004
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CD44 Alternative splicing

• The CD44 protein is a cell-surface glycoprotein
  involved in cell-cell interactions, cell adhesion
  and migration. It is a receptor for hyaluronic
  acid and can also interact with other ligands,
  such as osteopontin, collagens, and matrix
  metalloproteinases.
• This protein participates in a wide variety of
  cellular functions including lymphocyte
  activation, hematopoiesis, and tumor metastasis.
  
• Transcripts for this gene undergo complex
  alternative splicing that results in many
  functionally distinct isoforms

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Alternative splicing of CD44 pre-mRNA. Schematic
of the CD44 pre-mRNA, which has a variable
cassette exon cluster (v1v10), and the six
distinct mRNA transcripts (generated by
alternative splicing of the CD44 pre-mRNA) that
are known to be translated.
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Alternative splicing of CD45

• The protein encoded by this gene is a member of
  the protein tyrosine phosphatase (PTP) family.
  
• PTPs are known to be signaling molecules that
  regulate a variety of cellular processes
  including cell growth, differentiation, mitotic
  cycle, and oncogenic transformation.
• It is a type I transmembrane protein that is
  present in various forms on all differentiated
  hematopoietic cells that assists in the
  activation of those cells.
• The CD45 family consists of multiple members that
  are all products of a single complex gene.
  
• This gene contains 34 exons and three exons of
  the primary transcripts are alternatively spliced
  to generate up to eight different mature mRNAs
  and after translation eight different protein
  products

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Alternative splicing of CD45 pre-mRNA. Schematic
of the CD45 pre-mRNA, which contains 33 exons in
total. Exons 4, 5 and 6 are variable cassette
exons, which are alternatively spliced to give
rise to five mRNA transcripts (and resulting
protein isoforms). The expression of the various
exons is repressed after T-cell stimulation,
resulting in predominant expression of the CD45R0
isoform.
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Alternative Splicing in the Nervous System
AnEmerging Source of Diversity and Regulation

• C. J. Lee and K. Irizarry
• Biol Psychiatry 200354771776

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Different modes of alternative splicing and its
biological consequences. b) Alternative 3 splic
e-site usage, associated with differential use of
polyadenylation sites (represented by A) in the
vertebrate gene for calcitonin and
calcitonin-gene-related peptide (CGRP) generates
a calcium homeostatic hormone in the thyroid
gland or a vasodilator neuropeptide in the
nervous system. Processing patterns in green are
found in thyroid, those in red are found in
neurons.
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Impact of alternative splicing in the human
genome. Results of a genome-wide analysis of tiss
ue-specificity of alternative splice forms from E
ST data, showing the number of alternative splice
forms that were found to be specific to a single
tissue.
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Alterations of pre-mRNA splicing in cancer

• K. Z. Zayakin, P. Silina, and A. Line
• Genes Chromosomes Cancer. 2005 Apr42(4)342-57

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Genes Whose Splicing Pattern or Efficiency is
Altered in Cancer
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Multiple Alternative Splicing Markers for Ovarian
CancerKlinck et al. Cancer Res 2008 68
657-663

• Intense efforts are currently being directed
  toward profiling gene expression in the hope of
  developing better cancer markers and identifying
  potential drug targets.
• A sensitive new approach for the identification
  of cancer signatures based on direct
  high-throughput reverse transcription-PCR
  validation of alternative splicing events was
  developed.
• The splicing of 600 cancer-associated genes in 25
  normal and 21 serous ovarian cancer tissues was
  monitored.

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Results

• 4,700 alternative splicing events were screened,
  48 events were identified that were significantly
  associated with ovarian tumor tissues.
  
• In a further screen directed at 39 ovarian
  tissues containing cancer pathologies of various
  origins, this ovarian cancer splicing signature
  approach successfully distinguished all normal
  tissues from cancer.
• High-volume identification of cancer-associated
  splice forms by this procedure paves the way for
  the use of alternative splicing profiling to
  diagnose subtypes of cancer.

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SR Proteins in Alternative Splicing

• SR proteins have different RNA binding
  specificities.

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Role of SR Proteins in Alternative Splicing

• SR proteins can bind directly to weak 5 or 3
  splice sites to attract U1snRNP or U2AF
  
• SR proteins can bind to ESEs to attract
  spliceosome components to weak 5 or 3 splice
  sites positive regulation.
  
• SR proteins can compete with other RNA binding
  proteins called hnRNP proteins that block
  spliceosome formation -negative regulation.
  
• hnRNP proteins can bind to ESS (Exonic Splicing
  Silencer) sequences to prevent spliceosome
  formation.

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Sequences that bind to proteins that promote
spliceosome recognition of an exon are known as
exonic or intronic splicing enhancers (ESEs or
ISEs). Sequences that are required to inhibit rec
ognition of an exon are known as exonic or
intronic splicing silencers (ESSs or ISSs).
Two important conclusions from this model, which
hace been verified experimentally are
1. The mutation of sequences far from a splice
site can influence splicing. 2. The two states de
picted above are often in dynamic equilibrium,
such that subtle changes in the balance of ESE
and ESS-binding proteins can alter the ratio of
the mRNA isoform expression.
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Spinal Muscular Atrophy (SMA)

• Proximal spinal muscular atrophy (SMA) is an
  autosomal recessive disorder characterized by the
  degeneration of the motor neurons in the anterior
  horn of the spinal cord, resulting in muscular
  atrophy and weakness.
• The overall incidence of SMA is 1 in 10,000 live
  births, with a carrier frequency of 1 in 50.
  
• Onset is primarily in childhood, and three
  different forms are recognized type I SMA being
  the most severe form and type III SMA at the
  milder end of the scale.
• Children affected by SMA I never sit and usually
  die within the first year of life.

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Spinal Muscular Atrophy (SMA)

• Spinal muscular atrophy results from the lack of
  functional survival of motor neuron 1 gene
  (SMN1), even though all affected individuals
  carry a nearly identical, normal SMN2 gene.
• SMN2 is only partially active because a
  translationally silent, single-nucleotide
  difference in exon 7 causes exon skipping.
  

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SMN
Splicing of pre-mRNAs from the human spinal
muscular atrophy (SMA) survival of motor neuron 1
(SMN1) and SMN2 genes. In SMN1, exon 7 is a
constitutive exon, which harbors the
normal termination codon. In SMN2, exon 7 is
alternatively spliced 80 of the spliced
mRNAs skip exon 7, such that translation
terminates at exon 8. The resulting SMN2
protein has a different carboxy terminus, which
renders it unstable. Twenty per cent of the
SMN2-spliced mRNAs include exon 7, which gives
rise to full-length, functional SMN
protein. A single, translationally silent C ? T
transition at position 6 of exon 7 is
responsible for these splicing differences. This
nucleotide change inactivates an
SF2/ASF-dependent exonic splicing enhancer (ESE),
which begins at position 6.
SMA patients can produce functional SMN protein
but at levels that are insufficient for normal
function, which results in the progressive
degeneration of spinal-cord motor neurons.
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Disruption of an SF2/ASF-dependent exonic
splicing enhancer in SMN2 causes spinal muscular
atrophy in the absence of SMN1
Luca Cartegni Adrian R. Krainer
Nature Genetics volume 30 2002
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Exon 7skipping correlates with disruption of the
proximal SF2/ASF heptamer motif.
Semi-quantitative RTPCR analysis of transient
expression of SMN minigenes. The products
corresponding to exon 7skipping and inclusion
are indicated. The A11G suppressor mutation that
reconstitutes an SF2/ASF motif (lanes 4 and 6)
restores correct splicing when the mutation at
position 6 causes exon skipping (lanes 3 and 5).
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SF2/ASF binds directly to the SF2/ASF motif in
SMN1 exon 7
Phosphorimage scan of proteins crosslinked to
labeled RNA fragments, recovered by
immunoprecipitation with anti-SF2/ASF monoclonal
antibody and separated by SDSPAGE. The 42-nt RNA
probe was derived from SMN1 (lane 1) or SMN2
(lane 2).
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Activation of -Tropomyosin Exon 2 Is Regulated by
the SR Protein 9G8 and Heterogeneous Nuclear
Ribonucleoproteins H and F (hnRNP H and F)
J. Barrett Crawford and James G. Patton
MOLECULAR AND CELLULAR BIOLOGY, 2006, Vol. 26 8
7918802
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• The inclusion of exons 2 and 3 of a-
  tropomyosin is governed through tissue-specific
  alternative splicing. These exons are mutually
  exclusive, with exon 2 included in smooth muscle
  cells and exon 3 included in nearly all other
  cell types.
• Cis-acting sequences contribute to this splicing
  decision the branchpoints and pyrimidine tracts
  upstream of both exons and a series of
  purine-rich enhancers in exon 2.
• Here it is shown that a 35-kDa SR protein, 9G8,
  can activate the splicing of a-tropomyosin exon 2
  by binding ESEs.
  
• Further, hnRNP H and F bind to and compete for
  the same elements.
  
• Overexpression of hnRNPs H and F blocked
  9G8-mediated splicing both in vivo and in vitro,
  and siRNA-directed depletion of H and F led to an
  increase in exon 2 splicing.
• These data suggest that the activation of exon 2
  is dependent on the antagonistic activities of
  9G8 and hnRNPs H and F.

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(A) Exons 1 to 4 of -TM and splicing regulatory
elements are shown, encompassing the
branchpoint/pyrimidine tracts of exons 2 (B2P2)
and 3 (B3P3), upstream and downstream regulatory
elements (URE and DRE), and purine-rich enhancers
in exon 2 (denoted by vertical lines). A portion
of the exon 2 sequence is shown below, with the
four enhancers underlined, denoted B, A, X, and M.
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9G8 activates a-TM exon 2 inclusion.
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hnRNP H and hnRNP F antagonize 9G8-mediated
activation.
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hnRNPs H and F directly compete with 9G8 for
binding to the exon 2 enhancers.
(A) Radiolabeled wild-type -TM transcripts (25
nM) were incubated with 1 mM 9G8 in the presence
or absence of increasing amounts of hnRNPs H and
F. Reactions were subjected to UV cross-linking,
and labeled proteins were analyzed on 10 SDS
gels.
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siRNA depletion of hnRNP H and hnRNP F.
(A) Wildtype TM 1-4 minigene was cotransfected
into cells with siRNAs directed against hnRNP H
and/or hnRNP F, either alone, together, or in
combination with 9G8 expression plasmid. Splicing
patterns were analyzed. (B) Averages and standard
errors for exon 2 inclusion are shown.
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Conclusions

• The 35 kDa SR protein 9G8 was found to interact
  with purine-rich exonic enhancers in exon 2, and
  to stimulate exon 2 inclusionthat is, the smooth
  muscle splicing pattern.
• hnRNP H and F were found to bind to the same
  sequences and to antagonize 9G8 binding.
  
• This is consistent with a model of antagonism
  between 9G8 and hnRNP H and F for activation or
  repression of exon 2 splicing.
  
• Further studies will be done to determine tissue
  specific expression of 9G8 and hnRNP H/F.

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SRp38 Regulates Alternative Splicing and Is
Required for Ca2 Handling in the Embryonic Heart

• Y. Feng, M.T. Valley, J. Lazar, A.L. Yang, R.T.
  Bronson, S.Firestein, W.A. Coetzee, and J.L.
  Manley
  
• Developmental Cell 16, 528538, April 21, 2009

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Abstract

• SRp38 is an SR protein splicing regulator.
• To define the functions of SRp38 in vivo, SRp38
  null mice were generated.
  
• The majority of homozygous mutants survived only
  until E15.5 and displayed multiple cardiac
  defects.
  
• Evaluation of gene expression profiles in the
  SRp38/ embryonic heart revealed a defect in
  processing of the pre-mRNA encoding cardiac
  triadin, a protein that functions in regulation
  of Ca2 release from the sarcoplasmic reticulum
  during excitation-contraction coupling.
• This defect resulted in significantly reduced
  levels of triadin, as well as those of the
  interacting protein calsequestrin 2.
  
• Purified SRp38 was shown to bind specifically to
  the regulated exon and to modulate triadin
  splicing in vitro.
  
• Isolated SRp38/ embryonic cardiomyocytes
  displayed defects in Ca2 handling compared with
  wild-type controls.
  
• These results demonstrate that SRp38 regulates
  cardiac-specific alternative splicing of triadin
  pre-mRNA and is essential for proper Ca2
  handling during embryonic heart development.

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Figure 1. The Majority of SRp38/ Mice Die before
Birth and Display Severe Edema
(A) Mutant mice that survive to term are severely
growth retarded and die within the first day.
(B) Severe edema is visible in the E14.5
SRp38/embryo. The arrow indicates edema along the
back of the SRp38/ embryo.
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Splicing Patterns and Significantly Reduced
Triadin and CSQ2 Protein Levels Are Observed in
the SRp38/ Hearts (A) Triadin gene expression is
regulated by alternative RNA splicing. Exons 18
are shared by all three isoforms, while exon 9,
exon 10, and exon 11 are specific to triadin 1,
triadin 2, and triadin 3 mRNAs, respectively. (B)
Comparison of the levels of triadin transcript in
SRp38/ and SRp38/ hearts at E13.5 and E14.5.
Ventricular RNA was extracted from SRp38/ and
SRp38/ embryonic hearts and used for real-time
RT-PCR analysis. (C) Total triadin mRNA levels
(triadin 1 plus triadin 2) were reduced in SRp38/
hearts. Real-time RT-PCR was performed using
primers targeted against constitutively spliced
exons of triadin pre-mRNA. (D) CSQ2 mRNA levels
in SRp38/ and SRp38/ hearts. Real-time RT-PCR
was performed. (E) Decreased levels of triadin
and CSQ2 proteins in the SRp38/ heart. Ventricle
extracts were prepared from E14.5 embryos,
resolved by SDS-PAGE, and analyzed with indicated
antibodies.
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SRp38 Specifically Binds to Triadin-1-Specific
Exon 9 RNA (A) Schematic diagram of triadin const
itutive exon 8 (E8) and alternative exon 9 (E9).
Filled boxes denote two putative SRp38 binding
sites identified in E9. SS, splice site ASS,
alternative splice site. (B) Comparison of the
two putative SRp38 binding sites within E9 with
the selected SRp38 consensus sequence. (C) SRp38
binds to triadin 1-specific E9 RNA but not to
triadin 2-specific E10 RNA. In vitro transcribed
triadin 1-specific E9 or triadin 2-specific E10
RNA was used in a gel shift assay with increasing
amounts of GST-tagged SRp38 RBD as indicated. (D)
SRp38 only weakly binds to E9 mutant RNA. Two
SRp38 binding sites were replaced with the
indicated nucleotides in E9 RNA. Gel shift assays
were performed. (E) Competition assay with
32P-labeled E9 RNA and increasing amounts of
unlabeled RNAs as competitors. Gel shift assays
were performed. Each competitor was used at 2- or
6-fold molar excess over the probe. RNA-protein
complex was indicated by arrows
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SRp38 Specifically Activates Splicing of a
Substrate Containing Triadin-1-Specific Exon 9
RNA (A) SRp38 activates splicing of a substrate c
ontaining triadin 1-specific E9. In vitro
splicing was performed in HeLa S100 extract using
E9, E9 mutant, or E10 containing substrates
(b-E9, b-E9 mutant, or b-E10) with indicated
additions. SRp38 splicing activity in S100
requires a nuclear fraction (NF40-60) prepared by
ammonium sulfate precipitation. (B) Splicing of
b-E9 in vitro in the presence of unlabelled
competitors. Competitors were used at 3- or
9-fold molar excess over the pre-mRNA.
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The Incidence of Spontaneous Ca2 Sparks Is
Increased in SRp38/Myocytes (A) Confocal images o
f cardiomyocytes isolated from embryos at E14.5.
(B) Representative line-scan images of Ca2
sparks in myocytes incubated in Tyrodes solution
containing 5 mM Ca2. (C) Spark frequency was
compared between SRp38/ and SRp38/
cardiomyocytes. Comparison of distribution of
Ca2 sparks between SRp38/ and SRp38/
cardiomyocytes was made by plotting number of
sparks events against (D) spark amplitude (DF/F),
(E) full duration at half-maximal amplitude
(FDHM ms), or (F) full width at half-maximal
amplitude (FWHM mm), respectively. SparkMaster
was used to detect and analyze spark parameters.
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Conclusions

• During mouse development, the heart undergoes
  dramatic morphological changes and critical
  modifications in gene expression.
  
• Here, evidence was provided that the splicing
  factor SRp38 is critically involved in cardiac
  development and that its loss leads to multiple
  cardiac defects, changes in triadin pre-mRNA
  splicing, and altered intracellular Ca2 handling
  in isolated cardiomyocytes.
• SRp38 functions as an important regulator of
  alternative splicing during heart development and
  loss of SRp38 leads to changes in triadin
  pre-mRNA splicing and altered Ca2 handling in
  embryonic cardiomyocytes

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Alternative splicing

• Alternative splicing occurs in at least 60 of
  human genes
  
• Alternative splicing frequently occurs in a
  tissue specific or development specific manner
  
• Alternative splicing results from positive
  regulation by binding of SR proteins to ESEs or
  ISEs and from negative regulation by binding of
  hnRNP proteins to ESSs or ISSs.

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Splicing is regulated by cis-elements (ESE, ESS,
ISS, and ISE) and trans-acting splicing factors
(SR proteins, hnRNP, and unknown factors).