Novel RNAs along the Pathway of Gene Expression. (or, The Expanding Universe of Small RNAs)

Transcription

1 Novel RNAs along the Pathway of Gene Expression (or, The Expanding Universe of Small RNAs)

2 Central Dogma DNA RNA Protein replication transcription translation

3 Central Dogma DNA RNA Spliced RNA Protein replication transcription splicing translation

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5 ß Lupus and the Discovery of snrnps (pronounced snurps) ß Current Challenges in Splicing ß MicroRNAs: the latest novel RNAs in Gene Regulation

6 Immune System Antibodies normally against: bacteria viruses (cancer cells) abnormal against: own cellular components = Autoantibodies

7 Autoantibodies antibody cellular component immune complex

8 DNA RNA Protein

9 Amount of DNA in Phage, Bacterium, and Mammalian Cell

10 Nascent RNAs Are Coated with Proteins

11 Genes in Pieces exon intron exon intron exon Splicing messenger RNA

12 Introns Even though the 3-letter code remains wpsjkdmspsxmzpyrtgkslqrabkbdifqgpmwxoltfjvsydorwmbxlzfreualksdjhzxmt intact, many genes are interrupted by long segments of gibberish pjsgelvnyisgqzhbmsjq2qqndpqpoewirualsdfjzxcmvn\rlj DNA containing no 3-letter words specifying amino acids

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14 Autoantibodies antibody cellular component immune complex

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17 anti-ribosome anti-rnp

18 Base Pairing Interactions in Splicing

19 SnRNP Binding Determines Minimum Intron Size

20 Splicing in higher cells: an extra step in gene expression

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22 ATP ADP ATP ADP

23 ß Lupus and the Discovery of snrnps (pronounced snurps) ß Current Challenges in Splicing ßIs the spliceosome a ribozyme? ßHow is alternative splicing regulated? ßCoordination of splicing with other events in gene expression. ß MicroRNAs: the latest novel RNAs in

24 Human Spliceosomal snrnas

25 The Spliceosome: a Group II Intron in Pieces?

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30 Introns Are Larger than Exons J. Valcarcel

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32 COUPLING OF STEPS IN GENE EXPRESSION Maniatis & Reed (2002) Nature 416,

33 Nonsense Mediated mrna Decay (NMD) nucleus cytoplasm pre-mrna mrna Capping, splicing and polyadenylation 40S 60S AAAAAAAA TRANSLATION AAAAAAAAA Full-length Protein AAAAAAAAA mrna with a premature stop 40S 60S AAAAAAAA PREMATURE STOP DETECTED mrna decapped and degraded!

34 Nonsense-Mediated mrna Decay (NMD): mrna Surveillance Detects presence of a premature termination codon (PTC) arising from mutation or incomplete processing. Prevents synthesis of truncated, potentially deleterious proteins. Conserved in eukaryotes from yeast to man. Important for making disease genes recessive and for lymphocytes. Requires active translation. At least three factors Upf1, 2, and 3 involved.

35 An Exon Junction Complex (EJC) Is Deposited on the Spliced mrna by the Spliceosome EJC first described by LeHir, Izaurralde, Maquat, and Moore (2000) EMBO J. 19, Tang, Nott and Moore, Current Opinion in Cell Biology (2004) 16, 279.

36 Nonsense-Mediated mrna Decay (NMD) spliceosome spliceosome AUG UAA AAAAAAA No PTC AUG EJC UAA AAAAAAA Pioneer round of translation AUG UAA AAAAAAA Zhang and Krainer

37 Nonsense-Mediated mrna Decay (NMD) spliceosome spliceosome AUG UAA AAAAAAA No PTC With PTC AUG EJC UAA AAAAAAA AUG UAA EJC UAA AAAAAAA Pioneer round of translation Pioneer round of translation? AUG UAA AAAAAAA AUG UAA UAA AAAAAAA AUG UAA AAAAAAA AUG UAA UAA A A A A A A Multiple rounds of translation mrna degradation (from both 5 and 3 ends) Zhang & Krainer

38 ß Lupus and the Discovery of snrnps (pronounced snurps) ß Current Challenges in Splicing ß MicroRNAs: the latest novel RNAs in Gene Regulation

39 RNA Interference (RNAi) RNAi (RNA interference) was discovered when attempts at overexpression from transgenes in worms and plants led to no expression whatsoever! It was realized (by A. Fire and C. Mello) that dsrna triggers the degradation of complementary mrna. Thus, RNAi is a wonderful tool for functional gene knockouts. BUT, why is this machinery present in cells??? To process 100s of micrornas that function to regulate translation, transcription and even chromosome remodeling!

40 Cap... A (n)... Nuc. Cyt. AGO P P mirna gene P mirnp P Drosha RAN-GTP Exportin5 Dicer Primary transcript mirna precursor mirna:mirna* duplex Silencing complex P Transgene, transposon, heterochromatin, virus, etc. P AGO P P P RISC RdRP Dicer Dicer Dicer RISC P RISC P P P (sometimes) dsrna sirna duplexes Silencing complexes D. Bartel

41 Cap... A (n)... mirna gene Primary transcript Transgene, transposon, heterochromatin, virus, etc. dsrna mirnp RISC P Silencing complex RISC P Silencing complexes RISC P RISC P

42 All the Critical Steps in Eukaryotic Gene Expression Are Subject to Contol by RNA Silencing Pathways

43 RNA Silencing Pathways Are Ancient, Diverse and Essential

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46 Nonsense-mediated mrna decay (NMD) occurs when a ribosome stalls at a nonsense codon located upstream of an exon junction complex (EJC). CBC hnrnps Spliceosome hnrnps PABP-II pre-mrna 5 Exon 1 Exon 2 3 Nucleus mrna Cytoplasm hupf3 Decapping Intron Post Splicing Complex RNPS1 SRm160? REF/Aly 3 p15 TAP hgle2 hdbp5 DEK Y14 Nuclear Pore Complex hupf s EJC first described by LeHir et al. (2000) EMBO J.19, eif4f AUG mrna Ribosome RF PABP-I Premature termination

47 The Synthesis of Proteins Upon Ribosomes J.D. Watson (1964) Bull. Soc. Chim. Biol. 46,

48 Catalytic Cores of the Major and Minor Spliceosome

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50 COUPLING OF STEPS IN GENE EXPRESSION Maniatis & Reed (2002) Nature 416,