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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. Chen & Manley, 2009. Chen & Manley, 2009. How are different splicing pathways selected?.

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slide1

ALTERNATIVE SPLICING

Examples of pathways ...

Irimia & Blencowe Curr Opin Cell Biol 24:323, 2012

slide2

Types of alternative splicing vary in frequency among eukaryotes

Kim Nucl Acids Res 35: 125, 2006

slide3

Chen & Manley, 2009

Chen & Manley, 2009

How are different splicing pathways selected?

Splicing regulatory proteins assist or antagonize splice site recognition

Fu Cell 119: 736, 2004

slide4

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

slide5

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

slide6

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

slide7

How to discover new protein-protein interactions?

Yeast two-hybrid assay

Prp8p = U5 snRNP protein

Prp40p = U1 snRNP protein

Fused to transcription factor domain

of TF

In mammals:

Mud2p = U2AF65

& BBP = SF1

Alberts Fig. 5-81

Weaver, Fig. 14.37 &14.38

slide8

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

slide9

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

slide10

sxl

tra

dsx

Alberts Fig. 7.92

slide11

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

slide12

“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

slide13

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

slide14

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. PTB pyrimidine track binding protein)

If low pol II elongation rate (or internal pausing)…

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

slide17

High throughput technologies for analysis of alternative splicing

Hartman & Valcarcel Curr Opin Cell Biol 21:377, 2009

slide18

EST data mining & experimental verification

b) Microarray based detection

a) RT-PCR based detection

Modrek & Lee, Nat Genet 30:13, 2002

Wang & Cooper Nature Rev Genet. 8:749. 2007

slide19

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

slide21

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