RNA Interference and MicroRNA‐Mediated Silencing

Sylvia E.J. Fischer1

1 Departments of Genetics and Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 26.1
DOI:  10.1002/0471142727.mb2601s112
Online Posting Date:  October, 2015
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Abstract

RNA interference (RNAi) and microRNA‐mediated silencing cause sequence‐specific silencing of target genes. This overview will give a brief description of how RNAi and microRNAs were discovered, how small RNAs silence their targets, and what the functions of small RNAs are. Since the discovery of RNAi, RNAi has been widely used in studies of gene function, including high‐throughput screening. The unit will briefly describe how RNAi is used in different model systems, and how to analyze the function of endogenous small RNAs. © 2015 by John Wiley & Sons, Inc.

Keywords: RNAi; microRNA; silencing

     
 
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Table of Contents

  • The Discovery of Small RNA‐Mediated Gene Silencing
  • Small RNA‐Silencing Mechanisms
  • The Function of Small RNAs
  • RNAi in the Laboratory: Small RNA Analysis and Experimental Gene Silencing
  • Literature Cited
     
 
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Materials

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Literature Cited

Literature Cited
  Bagga, S., Bracht, J., Hunter, S., Massirer, K., Holtz, J., Eachus, R., and Pasquinelli, A.E. 2005. Regulation by let‐7 and lin‐4 miRNAs results in target mRNA degradation. Cell 122:553‐563.
  Bartel, D.P. 2009. MicroRNAs: Target recognition and regulatory functions. Cell 136:215‐233.
  Bernstein, E., Caudy, A.A., Hammond, S.M., and Hannon, G.J. 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363‐366.
  Burton, N.O., Burkhart, K.B., and Kennedy, S. 2011. Nuclear RNAi maintains heritable gene silencing in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 108:19683‐19688.
  Castel, S.E. and Martienssen, R.A. 2013. RNA interference in the nucleus: Roles for small RNAs in transcription, epigenetics and beyond. Nat. Rev. Genet. 14:100‐112.
  Catalanotto, C., Azzalin, G., Macino, G., and Cogoni, C. 2000. Gene silencing in worms and fungi. Nature 404:245.
  Cogoni, C. and Macino, G. 1999. Gene silencing in Neurospora crassa requires a protein homologous to RNA‐dependent RNA polymerase. Nature 399:166‐169.
  Dalmay, T., Hamilton, A., Rudd, S., Angell, S., and Baulcombe, D.C. 2000. An RNA‐dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101:543‐553.
  Ding, S.‐W. 2010. RNA‐based antiviral immunity. Nat. Rev. Immunol. 10:632‐644.
  Félix, M.‐A., Ashe, A., Piffaretti, J., Wu, G., Nuez, I., Bélicard, T., Jiang, Y., Zhao, G., Franz, C.J., Goldstein, L.D., Sanroman, M., Miska, E.A., and Wang, D.2011. Natural and experimental infection of Caenorhabditis nematodes by novel viruses related to nodaviruses. PLoS Biol. 9:e1000586.
  Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. 1998. Potent and specific genetic interference by double‐stranded RNA in Caenorhabditis elegans. Nature 391:806‐811.
  Giraldez, A.J., Mishima, Y., Rihel, J., Grocock, R.J., Van Dongen, S., Inoue, K., Enright, A.J., and Schier, A.F. 2006. Zebrafish MiR‐430 promotes deadenylation and clearance of maternal mRNAs. Science 312:75‐79.
  Grishok, A., Pasquinelli, A.E., Conte, D., Li, N., Parrish, S., Ha, I., Baillie, D.L., Fire, A., Ruvkun, G., and Mello, C.C. 2001. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106:23‐34.
  Guang, S., Bochner, A.F., Burkhart, K.B., Burton, N., Pavelec, D.M., and Kennedy, S. 2010. Small regulatory RNAs inhibit RNA polymerase II during the elongation phase of transcription. Nature 465:1097‐1101.
  Guo, S. and Kemphues, K J. 1995. par‐1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81:611‐620.
  Hamilton, A.J. and Baulcombe, D.C. 1999. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950‐952.
  Hammond, S.M., Bernstein, E., Beach, D., and Hannon, G.J. 2000. An RNA‐directed nuclease mediates post‐transcriptional gene silencing in Drosophila cells. Nature 404:293‐296.
  Hutvagner, G. 2001. A cellular function for the RNA‐interference enzyme Dicer in the maturation of the let‐7 small temporal RNA. Science 293:834‐838.
  Ketting, R.F. 2011. The many faces of RNAi. Dev. Cell 20:148‐161.
  Ketting, R.F., Haverkamp, T.H., van Luenen, H.G., and Plasterk, R.H. 1999. Mut‐7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD. Cell 99:133‐141.
  Ketting, R.F., Fischer, S.E., Bernstein, E., Sijen, T., Hannon, G.J., and Plasterk, R.H. 2001. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 15:2654‐2659.
  Kozomara, A. and Griffiths‐Jones, S. 2014. miRBase: Annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res. 42:D68‐D73.
  Lee, R.C., Feinbaum, R.L., and Ambros, V. 1993. The C. elegans heterochronic gene lin‐4 encodes small RNAs with antisense complementarity to lin‐14. Cell 75:843‐854.
  Maillard, P.V., Ciaudo, C., Marchais, A., Li, Y., Jay, F., Ding, S.W., and Voinnet, O. 2013. Antiviral RNA interference in mammalian cells. Science 342:235‐238.
  Montgomery, M.K., Xu, S., and Fire, A. 1998. RNA as a target of double‐stranded RNA‐mediated genetic interference in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 95:15502‐15507.
  Mourrain, P., Béclin, C., Elmayan, T., Feuerbach, F., Godon, C., Morel, J.B., Jouette, D., Lacombe, A.M., Nikic, S., Picault, N., Rémoué, K., Sanial, M., Vo, T.A., and Vaucheret, H. 2000. Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell 101:533‐542.
  Mukherji, S., Ebert, M.S., Zheng, G.X.Y., Tsang, J.S., Sharp, P.A., and van Oudenaarden, A. 2011. MicroRNAs can generate thresholds in target gene expression. Nat. Genet. 43:854‐859.
  Napoli, C., Lemieux, C., and Jorgensen, R. 1990. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co‐suppression of homologous genes in trans. Plant Cell Online 2:279‐289.
  Pasquinelli, A.E., Reinhart, B.J., Slack, F., Martindale, M.Q., Kuroda, M.I., Maller, B., Hayward, D.C., Ball, E.E., Degnan, B., Müller, P., Spring, J., Srinivasan, A., Fishman, M., Finnerty, J., Corbo, J., Levine, M., Leahy, P., Davidson, E., and Ruvkun, G. 2000. Conservation of the sequence and temporal expression of let‐7 heterochronic regulatory RNA. Nature 408:86‐89.
  Pumplin, N. and Voinnet, O. 2013. RNA silencing suppression by plant pathogens: Defense, counter‐defense and counter‐counter‐defense. Nat. Rev. Microbiol. 11:745‐760.
  Reinhart, B.J., Slack, F.J., Basson, M., Pasquinelli, A.E., Bettinger, J.C., Rougvie, A.E., Horvitz, H.R., and Ruvkun, G. 2000. The 21‐nucleotide let‐7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403:901‐906.
  Shi, Z., Hayes, G., and Ruvkun, G. 2013. Dual regulation of the lin‐14 target mRNA by the lin‐4 miRNA. PloS One 8:e75475.
  Sijen, T., Fleenor, J., Simmer, F., Thijssen, K.L., Parrish, S., Timmons, L., Plasterk, R.H., and Fire, A. 2001. On the role of RNA amplification in dsRNA‐triggered gene silencing. Cell 107:465‐476.
  Smardon, A., Spoerke, J.M., Stacey, S.C., Klein, M.E., Mackin, N., and Maine, E.M. 2000. EGO‐1 is related to RNA‐directed RNA polymerase and functions in germ‐line development and RNA interference in C. elegans. Curr. Biol. 10:169‐178.
  Tabara, H., Sarkissian, M., Kelly, W.G., Fleenor, J., Grishok, A., Timmons, L., Fire, A., and Mello, C.C. 1999. The rde‐1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99:123‐132.
  Tuschl, T., Zamore, P.D., Lehmann, R., Bartel, D.P., and Sharp, P.A. 1999. Targeted mRNA degradation by double‐stranded RNA in vitro. Genes Dev. 13:3191‐3197.
  van der Krol, A.R., Mur, L.A., Beld, M., Mol, J.N., and Stuitje, A.R. 1990. Flavonoid genes in petunia: Addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2:291‐299.
  Wightman, B., Ha, I., and Ruvkun, G. 1993. Posttranscriptional regulation of the heterochronic gene lin‐14 by lin‐4 mediates temporal pattern formation in C. elegans. Cell 75:855‐862.
  Zamore, P.D., Tuschl, T., Sharp, P.A., and Bartel, D.P. 2000. RNAi: Double‐stranded RNA directs the ATP‐dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25‐33.
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