Examples of pathways ...

ALTERNATIVE SPLICING

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

Kim Nucl Acids Res 35: 125, 2006

Types of alternative splicing vary in frequency among eukaryotes

How are different splicing pathways selected?

Fu Cell 119: 736, 2004

Splicing regulatory proteins assist or antagonize splice site recognition

Chen & Manley, 2009 Chen & Manley, 2009

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

Martinez-Contreras PLos Biol 4:e21, 2006

Looping-out model of splicing regulation

ABS & FBS = high affinity binding sites for hnRNP A/B (or hnRNP F/H)

How to discover new protein-protein interactions?

Alberts Fig. 5-81

Weaver, Fig. 14.37 &14.38

Fused to transcription factor domain

Yeast two-hybrid assay

In mammals: Mud2p = U2AF65 & BBP = SF1

of TF

Prp40p = U1 snRNP protein

Prp8p = U5 snRNP protein

“Alternative splicing changes protein isoforms (red half squares) by introducing new protein sequences (yellow) that are encoded by alternative exons.”

Examples of the impact of alternative splicing on cellular functions

Kelemen Gene 514:1, 2013

1. Sex determination in Drosophila mediated by alternative splicing

“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

Splicing regulatory factors

Sxl (sex-lethal)

Tra (transformer)

Transcription factors for sex-identity genesDsx (doublesex)

- 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:

Alberts Fig. 7.92

sxl

tra

dsx

“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”.

Matlin Nature Rev.Mol Cell Biol 6:386, 2005

Another example: HIV retrovirus tat alternative splicing

Show predicted results of gel mobility shift experiments.

exon 3 exon 3

hnRNP A1

Akker J Mol Endocrin 27: 123, 2002

Interpretation of these data?

“Action of SF2/ASF and hnRNP A1 in the selection of alternative splice sites”

D = distal 5’ splice siteP = proximal

Dose-dependent regulation

- splicing efficiency dependent on relative concentration (or activity)of SR “regulators” eg. phosphorylation state

- cooperative or antagonistic interactions between SR-type proteins

Control of splicing pathway choice?

Transcription rate

- if pausing of RNA pol II, opportunity for selection of different splice sites?

Transcriptional activators

- use of different promoters for a particular gene can generate different alternatively-spliced mRNAs

Models how this might be achieved?

- perhaps mediated by RNA pol II CTD?

Pawlicki & Steitz Trends Cell Biol 20:52, 2010

Effect of RNA pol II elongation rate on splicing pathway?

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

chance for including alternative exon (which otherwise would be skipped over)?

If high processivity…

and strong 3’ splice site outcompetes weak one?“simultaneous” presentation of both introns to splicing machinery

Kinetic model

Luco et al. Cell 144:16, 2011

Histone methylation status “read” by adaptor & recruits splicing factors (eg. PTB pyrimidine track binding protein)

Chromatin adaptor model

Integrated model for the control of alternative splicing

Luco et al. Cell 144:16, 2011

Wang et al. Nature 456:470, 2008

How many human genes exhibit alternative splicing?

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

High throughput technologies for analysis of alternative splicing

EST data mining & experimental verification

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

a) RT-PCR based detection b) Microarray based detection

Modrek & Lee, Nat Genet 30:13, 2002

Schmucker Cell 101: 671, 2000

Dscam = Drosophila axon guidance receptor

(immunoglobulin superfamily)

“potentially generate more than38,000 Dscam isoforms….

- homologue of human Down syndrome cell adhesion molecule

Red = N-terminal half of Ig2 domainBlue = N-terminal half of Ig3 domainGreen = entire Ig3 domain

- role in neuron development & synapse formation

McManus & Gravely Curr.Opin.Gen.Dev 21:373, 2011

Therapeutic approaches utilizing splicing

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

a) Oligonucleotide (loss of function)

b) Oligonucleotide (gain of function)

c) Trans-splicing

Antisense oligomer blocks access to splicing cis-element

Bifunctional oligomers with targeting (antisense) & “effector” domains

Provide wt exon with strong 3’ ss (so that defective exon is by-passed)