Intronic Splicing Regulatory Elements Gene Yeo, Salk Institute Array-based discovery from neural differentiation of human ES cells C. Carson Genome-wide comparative genomics J. Simon Nicole Coufal, Christian Carson, Alysson Muotri, Xiangdong Xu, Tiffany Liang, Rusty Gage Eric Van Nostrand, Tiffany Liang

Understand the rules to predict (cell-type, signal-induced specific) alternative splicing Alternative Constitutive

ACEScan[+] exons on UCSC browser Yeo, PNAS 05

NMDA receptor 1 Sorek, Sugnet, Yeo

Cell-type specific AS controlled by cis- elements and trans-factors GGGGG UAGG UC-rich; UCUU UGCAUG

Identification Of Intronic Splicing Regulatory Elements 1. Count conserved kmers and unconserved kmers 2. Derived a chi-square score for significantly enriched and conserved kmers 3. Clustered the kmers into motif families 161,730 exons 24.2 Mb exonic sequence 129 Mb intronic sequence

158 downstream motif clusters 156 upstream motif clusters Example of a downstream motif cluster: TGCATG,TGCATGA, ATGCATG, CTGCATG, TGCATGC, TGCATGT, TGCATGG, GTGCATG What are the properties of these motifs? Positional biases? Near alternatively spliced exons? Expression biases? Overlap known elements?

76% of downstream ISREs exhibit significant position biases 80% of upstream ISREs exhibit significant position biases I. ISREs have strong positional distribution biases New protocol for computing conserved positional bias, while controlling for background conservation

II. ISREs are enriched near alternative exons 30% downstream ISREs 56% upstream ISREs Some resemble known binding sites FOX1,2Enriched near muscle alternative exons (Ares) Nova1,2 MBNL PTB CELF

III. Genes containing ISRE-proximal exons are tissue-specific Expression biases (71% downstream ISREs, 81% upstream ISREs)

IV. ISREs overlap with Exonic Splicing Silencers ESEFairbrother WG, Yeh RF, Sharp PA, Burge CB (2002) ESSWang Z, Rolish ME, Yeo G, Tung V, Mawson M et al. (2004) ISEYeo G, Hoon S, Venkatesh B, Burge CB (2004) ESE, ESSZhang XH, Chasin LA (2004)

Like ESS, do ISREs affect Splice Site Choice ? Competing 5’ss and 3’ss reporter (Wang et al, Mol Cell, July, 2006)

Downstream ISREs suppress intron-proximal 5’ splice sites

Upstream ISREs suppress intron-proximal 3’ splice sites

Applications of ISREs: (1) splicing arrays --tissue specific alternative splicing? Sugnet et al. PLoS Comput Biol, 2006 skipped included skipped included TGCATG ACTAAC TTGGTT TGGTTT GCATG TCATTTT TTTCAT Enriched ISREs in downstream introns Enriched Depleted musclebrain

Applications of ISREs: (2) predicting RNA binding sites 1.Several proteins have been reported to affect their own alternative splicing (e.g. hnRNP A1, SRP20, SC35, TIA1, TIAR2, FOX2, PTB) 2.Evolutionarily conserved AS exons have high intronic conservation flanking the exon, resulting in algorithms that perform genomic predictions of alternative conserved exons 3.Alternative conserved exons are enriched in genes encoding RNA binding proteins and splicing factors

Applications of ISREs: (2) predicting RNA binding sites

ISREs are likely functional ISREs identified in mammals via comparative genomics. ISREs have positional biases, are enriched in tissue-specific genes, and overlap with ESS. ISREs alter splice site choice in vitro. Some ISREs resemble known sites of known alt splicing factors. A fraction of ISREs are proximal to alternative exons. ISREs can be utilized to analyze splicing-array data. ISREs can be utilized to identify autoregulated exons, and has other implications.

Alternative splicing differences in human embryonic stem cells versus neuronal progenitors hESCNP from hESC Endogeneous NP 1.Developed an algorithm to detect AS from exon arrays (REAP). 2.Verify AS events. 3.Identify ISREs proximal to AS events. 4.Identify RNA binding protein.

Exon arrays have probesets in every exon Simple representation Exon array, alternative splicing, gene expression Tiling arrays

REAP predictions agree with A)EST-verified alternative splicing B)ACEScan[+] exons

Confirming isoforms by RT-PCR

Discovery of ISREs proximal to ES/NP alternative exons

ISREs in ES/NP ongoing… REAP algorithm designed for exon array based detection of AS events REAP[+] exons correlate with EST-based and ACEScan[+] exons ISREs identified specific for ES/NP AS events FOX1/2 may regulate ES/NP-specific AS events

Alternative Constitutive Computational Modeling, Integration Cis-elements Association of RNA binding proteins to elements Stem cells, early neuronal differentiation Alternative splicing at the Crick-Jacobs Center, Salk Institute