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ALTERNATIVE SPLICING
Examples of pathways ...
Irimia & Blencowe Curr Opin Cell Biol 24:323, 2012
Types of alternative splicing vary in frequency among eukaryotes
Kim Nucl Acids Res 35: 125, 2006
How are different splicing pathways selected?
Chen & Manley, 2009
Chen & Manley, 2009
Splicing regulatory proteins assist or antagonize splice site recognition
Fu Cell 119: 736, 2004
Positive regulation of alternative splicing
- active selection of different splice site
- role of enhancers (ESE or ISE) which interact with SR-type “activators”
- usually < 200 nt from splice site (typically in downstream exon)
often Pu-rich (or Py-rich) motifs or simple repeats
Chen & Manley Nature Rev Mol Cell Biol 10:741, 2009
Negative regulation
- use of normal splice site “blocked”
- role of silencers (ESS or ISS)
- cis-elements within exon (or intron) which interact
with SR-type “repressors”
eg. tissue-specific SR protein might prevent binding of U2AF to pyrimidine tract
… or binding of U1 snRNP to downstream 5’splice site
Looping-out model of splicing regulation
ABS & FBS = high affinity binding
sites for hnRNP A/B (or hnRNP F/H)
Martinez-Contreras PLos Biol 4:e21, 2006
How to discover new protein-protein interactions?
Yeast two-hybrid assay
Prp8p = U5 snRNP protein
Prp40p = U1 snRNP protein
Fused to transcription
factor domain
Alberts Fig. 5-81
of TF
In mammals:
Mud2p = U2AF65
& BBP = SF1
Weaver, Fig. 14.37 &14.38
Examples of the impact of alternative splicing on cellular functions
“Alternative splicing changes protein isoforms (red half squares) by introducing new
protein sequences (yellow) that are encoded by alternative exons.”
Kelemen Gene 514:1, 2013
1. Sex determination in Drosophila mediated by alternative splicing
- splicing pathway controls whether fly will be male or female
- ratio of X chromosomes to autosomes triggers cascade of
alternative splicing pathways for genes encoding:
Splicing regulatory factors
Sxl (sex-lethal)
Tra (transformer)
Transcription factors for sex-identity genes
Dsx (doublesex)
“Repression of tra 3’splice site involves interaction of SXL with ISS
embedded in polypyrimidine tract and prevention of U2AF binding.”
Matlin Nature Rev.Mol Cell Biol 6:386, 2005
sxl
tra
dsx
Alberts Fig. 7.92
Another example: HIV retrovirus tat alternative splicing
hnRNP A1
exon 3
exon 3
“Inclusion of exon 3... is determined by the nuclear ratio of specific
hnRNP and SR proteins... Multimerization of hnRNP A1 from a high
affinity ESS can be sterically blocked by interaction of SF2/ASF
with ESE”.
Show predicted results of gel mobility shift experiments.
Matlin Nature Rev.Mol Cell Biol 6:386, 2005
“Action of SF2/ASF and hnRNP A1 in the selection of alternative splice sites”
D = distal 5’ splice site
P = proximal
Interpretation of these data?
Akker J Mol Endocrin 27: 123, 2002
Control of splicing pathway choice?
Dose-dependent regulation
- splicing efficiency dependent on relative concentration (or activity)
of SR “regulators”
eg. phosphorylation state
- perhaps mediated by RNA pol II CTD?
- cooperative or antagonistic interactions between SR-type proteins
Transcriptional activators
- use of different promoters for a particular gene can generate
different alternatively-spliced mRNAs
Models how this might be achieved?
Transcription rate
- if pausing of RNA pol II, opportunity for
selection of different splice sites?
Pawlicki & Steitz Trends Cell Biol 20:52, 2010
Effect of RNA pol II elongation rate on splicing pathway?
Kinetic model
Chromatin adaptor model
Histone methylation status “read” by
adaptor & recruits splicing factors (eg.
If low pol II elongation rate (or internal pausing)… PTB pyrimidine track binding protein)
chance for including alternative exon (which
otherwise would be skipped over)?
If high processivity…
“simultaneous” presentation of both introns to splicing machinery
and strong 3’ splice site outcompetes weak one?
Luco et al. Cell 144:16, 2011
Integrated model for the control of alternative splicing
Luco et al. Cell 144:16, 2011
How many human genes exhibit alternative splicing?
Wang et al. Nature 456:470, 2008
High throughput technologies for analysis of alternative splicing
Hartman & Valcarcel Curr Opin Cell Biol 21:377, 2009
EST data mining & experimental verification
a) RT-PCR based detection
b) Microarray based detection
Modrek & Lee, Nat Genet 30:13, 2002
Wang & Cooper Nature Rev Genet. 8:749. 2007
Dscam = Drosophila axon guidance receptor
- role in neuron development & synapse formation
- homologue of human Down
syndrome cell adhesion molecule
(immunoglobulin superfamily)
Red = N-terminal half of Ig2 domain
Blue = N-terminal half of Ig3 domain
Green = entire Ig3 domain
“potentially generate more than
38,000 Dscam isoforms….
Schmucker Cell 101: 671, 2000
McManus & Gravely Curr.Opin.Gen.Dev 21:373, 2011
Therapeutic approaches utilizing splicing
a) Oligonucleotide (loss of function)
Antisense oligomer blocks
access to splicing cis-element
b) Oligonucleotide (gain of function)
Bifunctional oligomers with
targeting (antisense) &
“effector” domains
c) Trans-splicing
Provide wt exon with strong 3’
ss (so that defective exon is
by-passed)
Wang & Cooper Nature Rev Genet. 8:749. 2007