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RNAi and Gene Silencing. Pete Burrows MIC 759 September 16, 2008. Lecture Outline. Background/discovery siRNA/shRNA Movie miRNA Biogenesis Functions Applications. # Publications. Focus on RNA interference - A user’s guide September 2006. 1998 Feb 19;391(6669):806-11 . No RNA.
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RNAi and Gene Silencing Pete Burrows MIC 759 September 16, 2008
Lecture Outline • Background/discovery • siRNA/shRNA • Movie • miRNA • Biogenesis • Functions • Applications
Focus on RNA interference - A user’s guide September 2006
1998 Feb 19;391(6669):806-11 No RNA No probe dsRNA anti-sense ssRNA In situ hybridization for mex-3 mRNA
Puzzles of the RNAi • Both sense and anti-sense ssRNA effective • Catalytic – very few copies of dsRNA could silence abundant mRNA • Therefore not conventional antisense • Only dsRNA targeting mature mRNA are effective, not to introns or promoters • RNAi can cross cellular boundaries
Classes of small RNAs dsRNA
RNAiDicing and slicing • All RNA silencing pathways are triggered by 21-27 nt long small RNAs • Small interfering RNAs – siRNA • Micro RNAs – miRNA • Piwi RNA • RNAi induction using long dsRNA only operates in plants and invertebrates • In mammals, long dsRNA (>30 bp) induces on the IFN response including PKR, inhibits translation, and activation of RNaseL, degrades mRNA
siRNA and shRNA • siRNA (short interfering RNA) • typically synthesized chemically then introduced into target cells • shRNA (short hairpin RNA) • typically introduced as a plasmid or viral vector • endogenous production, can be long term • enters the RNAi pathway upstream of siRNA
Dicer • Dicer generates RNAs with 2 nt 3’ overhang and 5’ phosphorylated terminus, both required for activity
RISC • RISC has helicase, endonucelase “slicer”,S and homology searching domains. • Initial RISC is inactive until transformed into active form by unwinding of the siRNA duplex and loss of sense strand.
Fig. 1. Only mammalian Ago2 can form cleavage-competent RISC Identification of Ago2 as “Slicer” in the RISC J. Liu et al., Science 305, 1437 -1441 (2004) Published by AAAS
Fig. 2. Argonaute2 is essential for mouse development J. Liu et al., Science 305, 1437 -1441 (2004) Published by AAAS
Fig. 3. Argonaute2 is essential for RNAi in MEFs J. Liu et al., Science 305, 1437 -1441 (2004) Published by AAAS
The ago1 mutant Arabidopsis develops abnormally because it does not produce an effector of silencing. The Argonaute genes were so named because the mutant plants look like an argonaute squid. The Sainsbury LaboratoryJohn Innes CentreColney LaneNorwich, NR4 7UH, UK
Summary of siRNA and shRNA processing
Processing of siRNA • Which becomes guide strand in the RISC and which is excluded? • Sequence and structure • Strand with the less-tightly base pared 5’ end is incorporated becomes guide strand
miRNA • Abundant ssRNA from a few thousand to 40,000 molecules /cell • Found in all metazoans • 0.5-1% of genes • siRNA targets genes from which it is derived in a sequence specific manner • miRNA regulates separate genes and has imperfect complementarity. May be 100’s/miRNA. Usually have many binding sites in each 3’ UTR, and several different miRNA can target same 3’ region. Combinatorial control • 30 – 50 % of genes regulated by miRNA
miRNA • Many miRNA are embedded in introns of protein encoding genes and are transcribed together with host genes. • miRNA can be expressed in developmentally tissue specific fashion but may not be expressed in tissues where putative target sequences are.
Tissue-specific expression of miRNA Plasterk RHA Cell 124:877 2006
The structure of human pri-miRNAs Du, T. et al. Development 2005;132:4645-4652
Processing of miRNA • Long primary Pol II transcript (pri-miRNA) • Cleaved by Drosha, nuclear RNase III endonuclease to establish one end of the miRNA (pre-miRNA) • Also need dsRNA binding protein Pasha (flies) DGCR8 (humans) • The pre-miRNA exported from the nucleus by Exportin 5 • Cut by Dicer→ miRNA • Strand with the less-tightly base pared 5’ end becomes mature miRNA, other strand becomes miRNA* and degraded • Worms and mammals only one Dicer and it makes miRNA and siRNA. Flies have one for each.
Imperfect homology between miRNA and 3’ UTR of target mRNA Seed sequence has perfect homology
Players in miRNA biogenesis • Drosha • Nuclear RNase III enzyme. Initiates miRAN biogenesis by cleaving pri-miRNA into pre-miRNA • Pasha • Partner of drosha is a dsRNA binding protein. Human DGCR8 • Exportin-5 • Nuclear transmembrane protein that transports pre-miRNA form nucleus to cytoplasm. Works in conjunction with GTP-Ran
Players in miRNA and siRNA • Argonaute (AGO) • PAZ domain binds the characteristic two-base 3' overhangs of siRNAs • PIWI domain: dsRNA guided hydrolysis of ssRNA • Slicer in RISC • Dicer (DCR) • Multi domain RNase III enzyme the cleaves dsRNA or stem-loop pre-miRNA into siRNA and miRNA • TRBP • Cofactor for Dicer • RISC • RNA induced silencing complex
Mechanism of miRNA suppression of gene expression • Transcription • mRNA degradation • Translational repression • 1 Initiation • 2 Post-initiation step • Co-translational degradation of the nascent peptide