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RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
RNA Silencing
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RNA Silencing

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  • siRNA artifacts?
  • ISH a. No probe, b. No RNA c. antisense RNA, d. dsRNA 4 cell C. elegans embryos
  • Cell transfected with myc tagged Ago plus siRNA for luciferase. Test IP’s for cleavage. Knew Argo part of RISC
  • C. Swelling of pericardium D. E. Failure to close neural tube
  • Traphina (Asci of the peach leaf curl fungus atop a peach leaf) sacchro, truffle, penicillium Fission yeast was isolated in 1893 by Lindner from East African millet beer . The species name is derived from the Swahili word for beer (Pombe). It was first developed as an experimental model for studying the cell cycle by Murdoch Mitchison in the 1950s. The fission yeast researcher Paul Nurse , together with Lee Hartwell and Tim Hunt , won the 2001 Nobel Prize in Physiology or Medicine , for their work on cell cycle regulation.
  • TRBP Tar RNA binding protein RCK human homolog of yeast Dhh 1p. Interacts with eIF4e and represses translation
  • Last one Phil Sharp
  • RT PCR olio dt and 3’UTR gene specific primer. Hinf1 digest of cDNA labeled 32P. Hinf1 site 95 bp from polyA site.
  • Full blown RISC 80S
  • Transcript

    • 1. RNA Silencing RNAi Post transcriptional gene regulation by siRNA and miRNA Pete Burrows MIC 759 October 26, 2006
    • 2. Small untranslated regulatory RNAs Eukaryotes ncRNA Prokaryotes sRNA
    • 3. Guillier, et al. Genes and Development 20:2338, 2006
    • 4.  
    • 5. Entrez PubMed Search Terms: RNAi or siRNA or miRNA Number of Publications
    • 6. Lecture Outline
      • Discovery
      • General features
      • siRNA
        • Role of translation
      • miRNA
      • Problems
    • 7. Focus on RNA interference - A user’s guide September 2006 Nature Genetics June 2006 Supplement
    • 8. Early demonstration of RNAi in plants
    • 9.  
    • 10.  
    • 11.  
    • 12. In situ hybridization for mex-3 mRNA 4 cell embryos No probe anti-sense ssRNA No RNA dsRNA 391:806, 1998
    • 13. Nature 391:744 1998 News and Views
    • 14. In vitro transcription Restriction digest Blunt end 5’ overhang 3’ overhang
    • 15. RNAi Dicing and slicing
      • RNA silencing pathways are triggered by 21-27 nt long small RNAs
        • Small interfering RNAs – siRNA
        • Repeat-associated small interfering RNAs –rasi RNAs
        • Micro RNAs – miRNA
        • Piwi-interacting RNA - piRNA
      • RNAi induction using long dsRNA only operates in plants and invertebrates
      • Worms – soak them in a solution of dsRNA, feed them bacteria expressing the appropriate construct
      • In vertebrates, long dsRNA (>30 bp) induces on the IFN response including PKR, inhibits translation, and activation of RNase L, degrades mRNA
    • 16. Novina and Sharp Nature 430:161 2004
    • 17.  
    • 18.  
    • 19. Dicer
      • Dicer generates RNAs with 2 nt 3’ overhang and 5’ phosphorylated terminus, both required for activity
      • Fly Dicer requires ATP, human may not
    • 20. RISC
      • RISC has helicase, exonuclease, endonucelase and homology searching proteins.
      • Initial RISC is inactive until transformed into active form by unwinding of the siRNA duplex and loss of sense (passenger) strand
      • Antisense (guide) strand defines specificity of RNAi
    • 21. Processing of siRNA
      • Starting with dsRNA
      • Which becomes guide strand in the RISC and which (passenger strand) is excluded?
        • Sequence and structure
        • Strand with the less-tightly base pared 5’ end is incorporated becomes guide strand
      • What is the endonuclease (Slicer) in RISC?
    • 22. 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 Laboratory John Innes Centre Colney Lane Norwich, NR4 7UH, UK Knew that Ago a RISC component
    • 23. Identification of Argonaute 2 as Slicer in humans Published by AAAS J. Liu et al., Science 305, 1437 -1441 (2004)
    • 24. Published by AAAS J. Liu et al., Science 305, 1437 -1441 (2004) Fig. 2. Argonaute2 is essential for mouse development
    • 25. Published by AAAS J. Liu et al., Science 305, 1437 -1441 (2004) Fig. 3. Argonaute2 is essential for RNAi in MEFs
    • 26. Sontheimer and Carthew, Science 2004 Sep 3;305(5689):1409-10
    • 27. Cytosolic players in siRNA and miRNA
      • Dicer ( DCR )
        • Multi domain RNase III enzyme the cleaves dsRNA or stem-loop pre-miRNA into siRNA and miRNA
      • TRBP
        • TAR RNA Binding Protein, Cofactor for Dicer
      • RISC
        • RNA induced silencing complex
      • Argonaute ( AGO )
        • PAZ domain binds the characteristic two-base 3' overhangs of siRNAs
        • PIWI domain: dsRNA guided hydrolysis of ssRNA
        • Ago2 is slicer in mammalian RISC
        • Other Ago may function in miRNA silencing
    • 28. Schizosaccharomyces pombe has DCR and AGO but not in S accharomyces cerevisiae Taphrina S. pombe S. cerevisiae Morel Penicillium http://www.glocalbeer.dk http://tolweb.org/tree?group=Ascomycota&contgroup=Fungi Swahili word for beer (Pombe)
    • 29. Is siRNA coupled to translation?
    • 30. Shen, et al. Differentiation 73:287-293 2005 Ferritin IRE-IRP
    • 31. Shen, et al. Differentiation 73:287-293 2005
    • 32. Shen, et al. Differentiation 73:287-293 2005
    • 33.  
    • 34. Fe + - + - + - + - ----Control---- -------IRE------- siRNA siRNA
    • 35. 7:719, 2005 7:633, 2005
    • 36. Colocalzation of Ago2 (Slicer) and Dcp1a (Decapping enzyme) in P-bodies
    • 37. Sen and Blau, The FASEB Journal, 2006 20:1293 RCK human homolog of yeast Dhh 1p Interacts with eIF4e and represses translation Targets RNA to P-bodies?
    • 38. RNAi The Movie Nature Genetics 2006
    • 39. miRNA
      • The miRNA are endogenous small RNA guides that repress the expression of target genes.
      • Differ from siRNA in biogenesis not in functions, although mechanisms can be different. mRNA cleavage when complementarity is extensive, repress translation when not.
      • lin-4 mutant worms had defects in timing of cell division. Encodes a small RNA that binds to and silenced lin-14 message.
      • Lin-14 mRNA levels do not decline, but that may not always be the case.
      • let-7 also found in other species.
    • 40. 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 regulate separate genes and has imperfect complementarity
      • May be 100’s mRNA regulated by one miRNA
      • Usually have many binding sites in each 3’ UTR, and several different miRNA can target same 3’ region. Combinatorial control
    • 41. 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.
    • 42. Plasterk Cell 124:877, 2006
    • 43. Du, T. et al. Development 2005;132:4645-4652 The structure of human pri-miRNAs
    • 44. 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.
    • 45. 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
    • 46. Cullen Nature Immunology 7:563 2006
    • 47. Du, T. et al. Development 2005;132:4645-4652 The miRNA biogenesis pathway
    • 48.  
    • 49. Mechanism of miRNA suppression of gene expression
      • Transcription
      • mRNA degradation
      • Translational repression
        • 1 Initiation
        • 2 Elongation
        • 3 Termination
        • 4 Release
      • Co-translational degradation of the nascent peptide
    • 50.  
    • 51.
      • Western blot for LIN-14 protein
      • Transcription same (run-on)
      • RNA levels ~ same
      • Lin-4 miRNA expressed at end of L1
    • 52. Measurement of lin-14 poly(A) tail length
    • 53. lin-14 RNase protection assay Polysome Profiles Polyribosome association of lin-14 mRNA is unchanged between L1 and L2
    • 54. lin-14 RNase protection assay Polysome Profiles EDTA sensitivity of polysomes
    • 55. Proc. Natl. Acad. Sci. USA 102: 16961, 2005
    • 56. Copyright ©2005 by the National Academy of Sciences Humphreys, David T. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 16961-16966 Fig. 1. miRNAs target the initiation step of translation No eIFs or Met-tRNA i met
    • 57. Fátima Gebauer & Matthias W. Hentze Nature Reviews Molecular Cell Biology 5, 827-835 (2004) Scanning model of Cap-dependent Translation Initiation E P A E P A eIF5B
    • 58. Other Studies
      • Both 5’ cap and 3’ poly(A) tail are necessary but not sufficient for miRNA repression of translation
      • EMCV IRES
        • Uses everything that canonical translation initiation does except eIF4E
        • Resistant to miRNA
      • New model
        • Block in translation initiation
        • Sequestration in P-bodies
        • In some cases this may lead to mRNA decay
    • 59. Is RNAi involved in viral immunity in the vertebrates?
      • Some viruses are more pathogenic in insects lacking dicer-2
      • Viruses encode proteins that inhibit Dicer
        • Flock house virus
        • Greasy grouper nervous necrosis virus
      • Does the host cell use RNAi to destroy viral RNA or inhibit its translation?
      • Predictions?
    • 60. Cullen Nature Genetics 38:S25, 2006
    • 61. In vivo applications of RNAi
      • Highly specific
        • Silence a single nucleotide difference in a dominant negative allele
      • Resistance not (less) a problem
        • Can design new RNAi if a mutation arises and original targeted sequence is changed
      • Problems
        • Stability
        • Delivery
        • Toxicity
    • 62. Off Target Effects
      • Global, due to induction of innate immune responses
      • Cross reactive, due to sequence homology with other mRNA sequences
      • Not easy to recognize unless global gene expression studies performed.
      • Good to have multiple target sequences
      • Recently found in flies
    • 63. Nature Biotechnology 24:697, 2006

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