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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. G m. AAAAA. G m. AAAAA.
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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?
Inferred Isoforms 0 10000 2000 4000 6000 8000 5970 Alternative isoforms 9527 All isoforms, including canonical 3557 genes with alternative spicing, from 12,000 mRNAs
Premature stop codon 2078 (35 % of 5970) Inferred Isoforms 0 10000 2000 4000 6000 8000 5970 Alternative isoforms 9527 All isoforms, including canonical
Nonsense-Mediated mRNA Decay Translated normally ORF Gm mRNA AAAAAAAAA Degraded by NMD ORF Gm mRNA AAAAAAAAA >50 nt
Alternative Splicing Can Yield Isoforms Differentially Subjected to Decay DNA DNA pre-mRNA pre-mRNA mRNA mRNA Nonsense mediated decay Premature termination codon
Eukaryota human, mouse, fly yeast
Eukaryote sequencing projects 1 3 6 1 16 1 8 5
The Players Upf1aka RENT1 (human), SMG-2 (C. elegans) Binds to downstream Upf2 and Upf3 during translation termination Promotes mRNA decapping and degradation Upf2aka SMG-3 (C. elegans) Binds to Upf3 after nuclear export Upf3aka SMG-4 (C. elegans) Associates with exon junction complex in nucleus
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%)
transcriptional regulation Gene locus transcription pre-mRNA productive splicing productive mRNA regulated unproductive splicing translation Protein
ORF SC35 pre-mRNA SC35 Auto-regulation Sureau et al. (2001) EMBO J20:1785-96
ORF SC35 pre-mRNA Gm AAAAA SC35 mRNA SC35 Auto-regulation Sureau et al. (2001) EMBO J20:1785-96
ORF SC35 pre-mRNA Gm AAAAA SC35 mRNA SC35 SC35 protein SC35 Auto-regulation Sureau et al. (2001) EMBO J20:1785-96
ORF SC35 pre-mRNA Gm AAAAA SC35 mRNA SC35 SC35 SC35 protein SC35 pre-mRNA SC35 Auto-regulation Sureau et al. (2001) EMBO J20:1785-96
ORF SC35 pre-mRNA Gm AAAAA SC35 mRNA SC35 SC35 SC35 protein SC35 pre-mRNA Gm AAAAA SC35 Auto-regulation SC35 mRNA (with premature termination codon) Sureau et al. (2001) EMBO J20:1785-96
ORF SC35 pre-mRNA Gm AAAAA SC35 mRNA SC35 SC35 SC35 protein SC35 pre-mRNA Gm AAAAA SC35 Auto-regulation SC35 mRNA (with premature termination codon) Sureau et al. (2001) EMBO J20:1785-96
SC35 Auto-regulation SC35 Locus transcription SC35 pre-mRNA alternative splicing splicing Productive SC35 mRNA translation SC35 protein