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Regulated Unproductive Splicing
Liana Lareau Brenner Group* *Labs aren’t carpeted. Groups are. Or so I’m told.
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Alternative Splicing Genomic DNA Sequence Transcription pre-mRNA
Intron Intron Intron Exon Exon Exon Exon Exon RNA Processing Gm AAAAA Gm AAAAA mRNA mRNA
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How much alternative splicing occurs in humans? What is its role?
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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
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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
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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
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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
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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
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Eukaryota
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Eukaryota human, mouse, fly yeast
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Eukaryote sequencing projects
1 6 3 1 16 1 5 8
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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
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Upf1
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Upf2
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Upf3
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Aspergillus, Neurospora
Eukaryota Plasmodium, Cryptosporidium human, mouse, fly, mosquito, worm yeast, S. pombe, Aspergillus, Neurospora
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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%)
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unproductive splicing
transcriptional regulation Gene locus transcription pre-mRNA productive splicing productive mRNA regulated unproductive splicing translation Protein
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SC35 Auto-regulation ORF SC35 pre-mRNA
Sureau et al. (2001) EMBO J 20:
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SC35 Auto-regulation ORF SC35 pre-mRNA SC35 mRNA Gm AAAAA
Sureau et al. (2001) EMBO J 20:
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SC35 Auto-regulation ORF SC35 pre-mRNA SC35 mRNA protein Gm AAAAA SC35
Sureau et al. (2001) EMBO J 20:
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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:
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(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:
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(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:
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SC35 Auto-regulation SC35 Locus SC35 pre-mRNA Productive SC35 mRNA
transcription SC35 pre-mRNA alternative splicing splicing Productive SC35 mRNA translation SC35 protein
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