Regulated Unproductive Splicing

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Regulated Unproductive Splicing Liana Lareau Brenner Group* *Labs aren’t carpeted. Groups are. Or so I’m told.

Alternative Splicing Genomic DNA Sequence Transcription pre-mRNA Intron Intron Intron Exon Exon Exon Exon Exon RNA Processing Gm AAAAA Gm AAAAA mRNA mRNA

How much alternative splicing occurs in humans? What is its role?

Alternative Isoform Inference TODO: Resolution, fonts, colors Show ResSeq-coding gene & how it maps to RefSeq For this & all slides, label items as DNA,pre-mRNA,mRNA,protein

Inferred Isoforms 2000 4000 6000 8000 10000 Alternative isoforms 5970 TODO: Update numbers yet again. All isoforms, including canonical 9527 3557 genes with alternative spicing, from 12,000 mRNAs

Inferred Isoforms 2000 4000 6000 8000 10000 Premature stop codon 2000 4000 6000 8000 10000 Premature stop codon 2078 (35 % of 5970) Alternative isoforms 5970 TODO: Update numbers yet again. All isoforms, including canonical 9527

Nonsense-Mediated mRNA Decay Translated normally ORF Gm mRNA AAAAAAAAA Degraded by NMD TODO: Add, for later display, 3rd case where additional intron had made stop codon premature. Need to show coding region for this Lollipop stop ORF Gm mRNA AAAAAAAAA >50 nt

Alternative Splicing Can Yield Isoforms Differentially Subjected to Decay DNA DNA pre-mRNA pre-mRNA mRNA TODO: Currently, two alternative splice forms in one nucleus. Bigger Label everything Better coloration Why is genome so funny shaped? Shadowing is excessive mRNA Nonsense mediated decay Premature termination codon

Eukaryota

Eukaryota human, mouse, fly yeast

Eukaryote sequencing projects 1 6 3 1 16 1 5 8

The Players Upf1 aka RENT1 (human), SMG-2 (C. elegans) Binds to downstream Upf2 and Upf3 during translation termination Promotes mRNA decapping and degradation Upf2 aka SMG-3 (C. elegans) Binds to Upf3 after nuclear export Upf3 aka SMG-4 (C. elegans) Associates with exon junction complex in nucleus

Upf1

Upf2

Upf3

Aspergillus, Neurospora Eukaryota Plasmodium, Cryptosporidium human, mouse, fly, mosquito, worm yeast, S. pombe, Aspergillus, Neurospora

Upf3: Yeast vs. Human BLAST results: Score = 26.9 bits (58), Expect = 9.8 Identities = 30/131 (22%), Positives = 48/131 (36%), Gaps = 16/131 (12%)

unproductive splicing transcriptional regulation Gene locus transcription pre-mRNA productive splicing productive mRNA regulated unproductive splicing translation Protein

SC35 Auto-regulation ORF SC35 pre-mRNA Sureau et al. (2001) EMBO J 20:1785-96

SC35 Auto-regulation ORF SC35 pre-mRNA SC35 mRNA Gm AAAAA Sureau et al. (2001) EMBO J 20:1785-96

SC35 Auto-regulation ORF SC35 pre-mRNA SC35 mRNA protein Gm AAAAA SC35 Sureau et al. (2001) EMBO J 20:1785-96

SC35 Auto-regulation ORF SC35 pre-mRNA SC35 mRNA protein SC35 pre-mRNA Gm SC35 mRNA AAAAA SC35 protein SC35 SC35 pre-mRNA Sureau et al. (2001) EMBO J 20:1785-96

(with premature termination codon) SC35 Auto-regulation SC35 pre-mRNA ORF Gm SC35 mRNA AAAAA SC35 protein SC35 SC35 pre-mRNA SC35 mRNA (with premature termination codon) Gm AAAAA Sureau et al. (2001) EMBO J 20:1785-96

(with premature termination codon) SC35 Auto-regulation SC35 pre-mRNA ORF Gm SC35 mRNA AAAAA SC35 protein SC35 SC35 pre-mRNA SC35 mRNA (with premature termination codon) Gm AAAAA Sureau et al. (2001) EMBO J 20:1785-96

SC35 Auto-regulation SC35 Locus SC35 pre-mRNA Productive SC35 mRNA transcription SC35 pre-mRNA alternative splicing splicing Productive SC35 mRNA translation SC35 protein