RNA Silencing RNAi Post transcriptional gene regulation by siRNA and miRNA Pete Burrows MIC 759 October 26, 2006
Small untranslated regulatory RNAs Eukaryotes ncRNA Prokaryotes sRNA
Guillier, et al.  Genes and Development  20:2338, 2006
 
Entrez PubMed Search Terms: RNAi or siRNA or miRNA Number of Publications
Lecture Outline <ul><li>Discovery </li></ul><ul><li>General features </li></ul><ul><li>siRNA </li></ul><ul><ul><li>Role of...
Focus on RNA interference  - A user’s guide September 2006 Nature Genetics June 2006 Supplement
Early demonstration of RNAi in plants
 
 
 
In situ  hybridization for  mex-3  mRNA 4 cell embryos No probe anti-sense ssRNA No RNA dsRNA 391:806, 1998
Nature 391:744 1998 News and Views
In vitro transcription Restriction digest Blunt end 5’ overhang 3’ overhang
RNAi Dicing and slicing <ul><li>RNA silencing pathways are triggered by 21-27 nt long small RNAs </li></ul><ul><ul><li>Sma...
Novina and Sharp  Nature  430:161 2004
 
 
Dicer <ul><li>Dicer generates RNAs with 2 nt 3’ overhang and 5’ phosphorylated terminus, both required for activity </li><...
RISC <ul><li>RISC has helicase, exonuclease, endonucelase and homology searching proteins. </li></ul><ul><li>Initial RISC ...
Processing of siRNA <ul><li>Starting with dsRNA </li></ul><ul><li>Which becomes guide strand in the RISC and which (passen...
The  ago1  mutant  Arabidopsis  develops abnormally because it does not produce an effector of silencing. The  Argonaute  ...
Identification of Argonaute 2 as Slicer in humans Published by AAAS J.  Liu et al.,  Science  305, 1437 -1441 (2004)
Published by AAAS J.  Liu et al.,  Science  305, 1437 -1441 (2004)  Fig. 2. Argonaute2 is essential for mouse development
Published by AAAS J.  Liu et al.,  Science  305, 1437 -1441 (2004)  Fig. 3. Argonaute2 is essential for RNAi in MEFs
Sontheimer and Carthew, Science 2004 Sep 3;305(5689):1409-10
Cytosolic players in siRNA and miRNA <ul><li>Dicer ( DCR ) </li></ul><ul><ul><li>Multi domain RNase III enzyme the cleaves...
Schizosaccharomyces pombe  has DCR and AGO but not in  S accharomyces  cerevisiae Taphrina   S. pombe   S. cerevisiae   Mo...
Is siRNA coupled to translation?
Shen, et al.  Differentiation  73:287-293 2005   Ferritin IRE-IRP
Shen, et al.  Differentiation  73:287-293 2005
Shen, et al.  Differentiation  73:287-293 2005
 
Fe  +  -  +  -  +  -  +  - ----Control---- -------IRE------- siRNA siRNA
7:719, 2005 7:633, 2005
Colocalzation of Ago2 (Slicer) and Dcp1a (Decapping enzyme) in P-bodies
Sen and Blau, The FASEB Journal, 2006 20:1293 RCK  human homolog of yeast Dhh 1p Interacts with eIF4e and represses transl...
RNAi The Movie Nature Genetics 2006
miRNA <ul><li>The miRNA are endogenous small RNA guides that repress the expression of target genes. </li></ul><ul><li>Dif...
miRNA <ul><li>Abundant ssRNA from a few thousand to 40,000 molecules /cell </li></ul><ul><li>Found in all metazoans </li><...
miRNA <ul><li>Many miRNA are embedded in introns of protein encoding genes and are transcribed together with host genes. <...
Plasterk  Cell  124:877, 2006
Du, T. et al. Development 2005;132:4645-4652 The structure of human pri-miRNAs
Processing of miRNA <ul><li>Long primary Pol II transcript (pri-miRNA) </li></ul><ul><li>Cleaved by Drosha, nuclear RNase ...
Players in miRNA biogenesis <ul><li>Drosha </li></ul><ul><ul><li>Nuclear RNase III enzyme. Initiates miRAN biogenesis by c...
Cullen  Nature Immunology 7:563 2006
Du, T. et al. Development 2005;132:4645-4652 The miRNA biogenesis pathway
 
Mechanism of miRNA suppression of gene expression <ul><li>Transcription </li></ul><ul><li>mRNA degradation </li></ul><ul><...
 
<ul><li>Western blot for LIN-14 protein </li></ul><ul><li>Transcription same (run-on) </li></ul><ul><li>RNA levels ~ same ...
Measurement of  lin-14  poly(A) tail length
lin-14  RNase protection assay Polysome Profiles Polyribosome association of lin-14 mRNA is unchanged between L1 and L2
lin-14  RNase protection assay Polysome Profiles EDTA sensitivity of polysomes
Proc. Natl. Acad. Sci. USA 102: 16961, 2005
Copyright ©2005 by the National Academy of Sciences Humphreys, David T. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 1696...
Fátima Gebauer & Matthias W. Hentze Nature Reviews Molecular Cell Biology  5, 827-835 (2004) Scanning model of Cap-depende...
Other Studies <ul><li>Both 5’ cap and 3’ poly(A) tail are necessary but not sufficient for miRNA repression of translation...
Is RNAi involved in viral immunity in the vertebrates? <ul><li>Some viruses are more pathogenic in insects lacking  dicer-...
Cullen  Nature Genetics  38:S25, 2006
In vivo  applications of RNAi <ul><li>Highly specific </li></ul><ul><ul><li>Silence a single nucleotide difference in a do...
Off Target Effects <ul><li>Global, due to induction of innate immune responses </li></ul><ul><li>Cross reactive, due to se...
Nature Biotechnology 24:697, 2006
Upcoming SlideShare
Loading in...5
×

RNA Silencing

1,735

Published on

Published in: Technology
0 Comments
2 Likes
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total Views
1,735
On Slideshare
0
From Embeds
0
Number of Embeds
0
Actions
Shares
0
Downloads
142
Comments
0
Likes
2
Embeds 0
No embeds

No notes for slide
  • 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
  • RNA Silencing

    1. 1. RNA Silencing RNAi Post transcriptional gene regulation by siRNA and miRNA Pete Burrows MIC 759 October 26, 2006
    2. 2. Small untranslated regulatory RNAs Eukaryotes ncRNA Prokaryotes sRNA
    3. 3. Guillier, et al. Genes and Development 20:2338, 2006
    4. 5. Entrez PubMed Search Terms: RNAi or siRNA or miRNA Number of Publications
    5. 6. Lecture Outline <ul><li>Discovery </li></ul><ul><li>General features </li></ul><ul><li>siRNA </li></ul><ul><ul><li>Role of translation </li></ul></ul><ul><li>miRNA </li></ul><ul><li>Problems </li></ul>
    6. 7. Focus on RNA interference - A user’s guide September 2006 Nature Genetics June 2006 Supplement
    7. 8. Early demonstration of RNAi in plants
    8. 12. In situ hybridization for mex-3 mRNA 4 cell embryos No probe anti-sense ssRNA No RNA dsRNA 391:806, 1998
    9. 13. Nature 391:744 1998 News and Views
    10. 14. In vitro transcription Restriction digest Blunt end 5’ overhang 3’ overhang
    11. 15. RNAi Dicing and slicing <ul><li>RNA silencing pathways are triggered by 21-27 nt long small RNAs </li></ul><ul><ul><li>Small interfering RNAs – siRNA </li></ul></ul><ul><ul><li>Repeat-associated small interfering RNAs –rasi RNAs </li></ul></ul><ul><ul><li>Micro RNAs – miRNA </li></ul></ul><ul><ul><li>Piwi-interacting RNA - piRNA </li></ul></ul><ul><li>RNAi induction using long dsRNA only operates in plants and invertebrates </li></ul><ul><li>Worms – soak them in a solution of dsRNA, feed them bacteria expressing the appropriate construct </li></ul><ul><li>In vertebrates, long dsRNA (>30 bp) induces on the IFN response including PKR, inhibits translation, and activation of RNase L, degrades mRNA </li></ul>
    12. 16. Novina and Sharp Nature 430:161 2004
    13. 19. Dicer <ul><li>Dicer generates RNAs with 2 nt 3’ overhang and 5’ phosphorylated terminus, both required for activity </li></ul><ul><li>Fly Dicer requires ATP, human may not </li></ul>
    14. 20. RISC <ul><li>RISC has helicase, exonuclease, endonucelase and homology searching proteins. </li></ul><ul><li>Initial RISC is inactive until transformed into active form by unwinding of the siRNA duplex and loss of sense (passenger) strand </li></ul><ul><li>Antisense (guide) strand defines specificity of RNAi </li></ul>
    15. 21. Processing of siRNA <ul><li>Starting with dsRNA </li></ul><ul><li>Which becomes guide strand in the RISC and which (passenger strand) is excluded? </li></ul><ul><ul><li>Sequence and structure </li></ul></ul><ul><ul><li>Strand with the less-tightly base pared 5’ end is incorporated becomes guide strand </li></ul></ul><ul><li>What is the endonuclease (Slicer) in RISC? </li></ul>
    16. 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
    17. 23. Identification of Argonaute 2 as Slicer in humans Published by AAAS J. Liu et al., Science 305, 1437 -1441 (2004)
    18. 24. Published by AAAS J. Liu et al., Science 305, 1437 -1441 (2004) Fig. 2. Argonaute2 is essential for mouse development
    19. 25. Published by AAAS J. Liu et al., Science 305, 1437 -1441 (2004) Fig. 3. Argonaute2 is essential for RNAi in MEFs
    20. 26. Sontheimer and Carthew, Science 2004 Sep 3;305(5689):1409-10
    21. 27. Cytosolic players in siRNA and miRNA <ul><li>Dicer ( DCR ) </li></ul><ul><ul><li>Multi domain RNase III enzyme the cleaves dsRNA or stem-loop pre-miRNA into siRNA and miRNA </li></ul></ul><ul><li>TRBP </li></ul><ul><ul><li>TAR RNA Binding Protein, Cofactor for Dicer </li></ul></ul><ul><li>RISC </li></ul><ul><ul><li>RNA induced silencing complex </li></ul></ul><ul><li>Argonaute ( AGO ) </li></ul><ul><ul><li>PAZ domain binds the characteristic two-base 3' overhangs of siRNAs </li></ul></ul><ul><ul><li>PIWI domain: dsRNA guided hydrolysis of ssRNA </li></ul></ul><ul><ul><li>Ago2 is slicer in mammalian RISC </li></ul></ul><ul><ul><li>Other Ago may function in miRNA silencing </li></ul></ul>
    22. 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)
    23. 29. Is siRNA coupled to translation?
    24. 30. Shen, et al. Differentiation 73:287-293 2005 Ferritin IRE-IRP
    25. 31. Shen, et al. Differentiation 73:287-293 2005
    26. 32. Shen, et al. Differentiation 73:287-293 2005
    27. 34. Fe + - + - + - + - ----Control---- -------IRE------- siRNA siRNA
    28. 35. 7:719, 2005 7:633, 2005
    29. 36. Colocalzation of Ago2 (Slicer) and Dcp1a (Decapping enzyme) in P-bodies
    30. 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?
    31. 38. RNAi The Movie Nature Genetics 2006
    32. 39. miRNA <ul><li>The miRNA are endogenous small RNA guides that repress the expression of target genes. </li></ul><ul><li>Differ from siRNA in biogenesis not in functions, although mechanisms can be different. mRNA cleavage when complementarity is extensive, repress translation when not. </li></ul><ul><li>lin-4 mutant worms had defects in timing of cell division. Encodes a small RNA that binds to and silenced lin-14 message. </li></ul><ul><li>Lin-14 mRNA levels do not decline, but that may not always be the case. </li></ul><ul><li>let-7 also found in other species. </li></ul>
    33. 40. miRNA <ul><li>Abundant ssRNA from a few thousand to 40,000 molecules /cell </li></ul><ul><li>Found in all metazoans </li></ul><ul><li>0.5-1% of genes </li></ul><ul><li>siRNA targets genes from which it is derived in a sequence specific manner </li></ul><ul><li>miRNA regulate separate genes and has imperfect complementarity </li></ul><ul><li>May be 100’s mRNA regulated by one miRNA </li></ul><ul><li>Usually have many binding sites in each 3’ UTR, and several different miRNA can target same 3’ region. Combinatorial control </li></ul>
    34. 41. miRNA <ul><li>Many miRNA are embedded in introns of protein encoding genes and are transcribed together with host genes. </li></ul><ul><li>miRNA can be expressed in developmentally tissue specific fashion but may not be expressed in tissues where putative target sequences are. </li></ul>
    35. 42. Plasterk Cell 124:877, 2006
    36. 43. Du, T. et al. Development 2005;132:4645-4652 The structure of human pri-miRNAs
    37. 44. Processing of miRNA <ul><li>Long primary Pol II transcript (pri-miRNA) </li></ul><ul><li>Cleaved by Drosha, nuclear RNase III endonuclease to establish one end of the miRNA (pre-miRNA) </li></ul><ul><ul><li>Also need dsRNA binding protein Pasha (flies) DGCR8 (humans) </li></ul></ul><ul><li>The pre-miRNA exported from the nucleus by Exportin 5 </li></ul><ul><li>Cut by Dicer -> miRNA </li></ul><ul><li>Strand with the less-tightly base pared 5’ end becomes mature miRNA, other strand becomes miRNA* and degraded </li></ul><ul><li>Worms and mammals only one Dicer and it makes miRNA and siRNA. Flies have one for each. </li></ul>
    38. 45. Players in miRNA biogenesis <ul><li>Drosha </li></ul><ul><ul><li>Nuclear RNase III enzyme. Initiates miRAN biogenesis by cleaving pri-miRNA into pre-miRNA </li></ul></ul><ul><li>Pasha </li></ul><ul><ul><li>Partner of drosha is a dsRNA binding protein. Human DGCR8 </li></ul></ul><ul><li>Exportin-5 </li></ul><ul><ul><li>Nuclear transmembrane protein that transports pre-miRNA form nucleus to cytoplasm. Works in conjunction with GTP-Ran </li></ul></ul>
    39. 46. Cullen Nature Immunology 7:563 2006
    40. 47. Du, T. et al. Development 2005;132:4645-4652 The miRNA biogenesis pathway
    41. 49. Mechanism of miRNA suppression of gene expression <ul><li>Transcription </li></ul><ul><li>mRNA degradation </li></ul><ul><li>Translational repression </li></ul><ul><ul><li>1 Initiation </li></ul></ul><ul><ul><li>2 Elongation </li></ul></ul><ul><ul><li>3 Termination </li></ul></ul><ul><ul><li>4 Release </li></ul></ul><ul><li>Co-translational degradation of the nascent peptide </li></ul>
    42. 51. <ul><li>Western blot for LIN-14 protein </li></ul><ul><li>Transcription same (run-on) </li></ul><ul><li>RNA levels ~ same </li></ul><ul><li>Lin-4 miRNA expressed at end of L1 </li></ul>
    43. 52. Measurement of lin-14 poly(A) tail length
    44. 53. lin-14 RNase protection assay Polysome Profiles Polyribosome association of lin-14 mRNA is unchanged between L1 and L2
    45. 54. lin-14 RNase protection assay Polysome Profiles EDTA sensitivity of polysomes
    46. 55. Proc. Natl. Acad. Sci. USA 102: 16961, 2005
    47. 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
    48. 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
    49. 58. Other Studies <ul><li>Both 5’ cap and 3’ poly(A) tail are necessary but not sufficient for miRNA repression of translation </li></ul><ul><li>EMCV IRES </li></ul><ul><ul><li>Uses everything that canonical translation initiation does except eIF4E </li></ul></ul><ul><ul><li>Resistant to miRNA </li></ul></ul><ul><li>New model </li></ul><ul><ul><li>Block in translation initiation </li></ul></ul><ul><ul><li>Sequestration in P-bodies </li></ul></ul><ul><ul><li>In some cases this may lead to mRNA decay </li></ul></ul>
    50. 59. Is RNAi involved in viral immunity in the vertebrates? <ul><li>Some viruses are more pathogenic in insects lacking dicer-2 </li></ul><ul><li>Viruses encode proteins that inhibit Dicer </li></ul><ul><ul><li>Flock house virus </li></ul></ul><ul><ul><li>Greasy grouper nervous necrosis virus </li></ul></ul><ul><li>Does the host cell use RNAi to destroy viral RNA or inhibit its translation? </li></ul><ul><li>Predictions? </li></ul>
    51. 60. Cullen Nature Genetics 38:S25, 2006
    52. 61. In vivo applications of RNAi <ul><li>Highly specific </li></ul><ul><ul><li>Silence a single nucleotide difference in a dominant negative allele </li></ul></ul><ul><li>Resistance not (less) a problem </li></ul><ul><ul><li>Can design new RNAi if a mutation arises and original targeted sequence is changed </li></ul></ul><ul><li>Problems </li></ul><ul><ul><li>Stability </li></ul></ul><ul><ul><li>Delivery </li></ul></ul><ul><ul><li>Toxicity </li></ul></ul>
    53. 62. Off Target Effects <ul><li>Global, due to induction of innate immune responses </li></ul><ul><li>Cross reactive, due to sequence homology with other mRNA sequences </li></ul><ul><li>Not easy to recognize unless global gene expression studies performed. </li></ul><ul><li>Good to have multiple target sequences </li></ul><ul><li>Recently found in flies </li></ul>
    54. 63. Nature Biotechnology 24:697, 2006
    1. A particular slide catching your eye?

      Clipping is a handy way to collect important slides you want to go back to later.

    ×