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Piecing the Puzzle Together: Genetic Requirements for miRNA Biogenesis in Arabidopsis thaliana

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Book cover Plant MicroRNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 592))

Abstract

MicroRNAs (miRNAs) are an important class of endogenous small silencing RNAs in both plants and animals. They regulate the expression of a wide range of target genes that are involved in many important biological processes. Biogenesis of plant miRNAs requires a distinct set of proteins, including members that belong to several highly conserved RNA silencing protein families. The framework for miRNA biogenesis in plants was revealed through genetic and biochemical analyses using mutants that are defective in miRNA accumulation. These general miRNA-deficient mutants constitute a set of invaluable genetic resources for the plant miRNA research community. They could be utilized to experimentally validate the candidate miRNAs that are either predicted by a computational program or recovered from a small RNA deep sequencing effort which is becoming a more affordable and widely used approach for small RNA discovery. Starting with a brief introduction on multiple small RNA pathways in plants, this chapter provides basic experimental procedures for the examination of miRNA accumulation from wild type plants and various mutant lines in Arabidopsis.

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References

  1. Llave C, Kasschau KD, Rector MA, Carrington JC (2002) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14: 1605–1619

    Article  CAS  PubMed  Google Scholar 

  2. Mette MF, van der Winden J, Matzke M, Matzke AJ (2002) Short RNAs can identify new candidate transposable element families in Arabidopsis. Plant Physiol 130:6–9

    Article  CAS  PubMed  Google Scholar 

  3. Park W, Li J, Song R, Messing J, Chen X (2002) CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr Biol 12:1484–1495

    Article  CAS  PubMed  Google Scholar 

  4. Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants. Genes Dev 16:1616–1626

    Article  CAS  PubMed  Google Scholar 

  5. Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950–952

    Article  CAS  PubMed  Google Scholar 

  6. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T (2001) Identification of novel genes coding for small expressed RNAs. Science 294:853–858

    Article  CAS  PubMed  Google Scholar 

  7. Lau NC, Lim EP, Weinstein EG, Bartel DP (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:858–862

    Article  CAS  PubMed  Google Scholar 

  8. Lee RC, Ambros V (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294:862–864

    Article  CAS  PubMed  Google Scholar 

  9. Baulcombe D (2004) RNA silencing in plants. Nature 431:356–363

    Article  CAS  PubMed  Google Scholar 

  10. Chapman EJ, Carrington JC (2007) Specialization and evolution of endogenous small RNA pathways. Nat Rev Genet 8:884–896

    Article  CAS  PubMed  Google Scholar 

  11. Jones-Rhoades MW, Bartel DP, Bartel B (2006) microRNAs and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53

    Article  CAS  PubMed  Google Scholar 

  12. Vaucheret H (2006) Post-transcriptional small RNA pathways in plants: mechanisms and regulations. Genes Dev 20:759–771

    Article  CAS  PubMed  Google Scholar 

  13. Axtell MJ, Snyder JA, Bartel DP (2007) Common functions for diverse small RNAs of land plants. Plant Cell 19(6):1750–1769

    Article  CAS  PubMed  Google Scholar 

  14. Fahlgren N, Howell MD, Kasschau KD, Chapman EJ, Sullivan CM, Cumbie JS, Givan SA, Law TF, Grant SR, Dangl JL, Carrington JC (2007) High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PLoS ONE 2:e219

    Article  PubMed  Google Scholar 

  15. Henderson IR, Zhang X, Lu C, Johnson L, Meyers BC, Green PJ, Jacobsen SE (2006) Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat Genet 38:721–725

    Article  CAS  PubMed  Google Scholar 

  16. Lu C, Tej SS, Luo S, Haudenschild CD, Meyers BC, Green PJ (2005) Elucidation of the small RNA component of the transcriptome. Science 309:1567–1569

    Article  CAS  PubMed  Google Scholar 

  17. Rajagopalan R, Vaucheret H, Trejo J, Bartel DP (2006) A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev 20:3407–3425

    Article  CAS  PubMed  Google Scholar 

  18. Xie Z, Qi X (2008) Diverse small RNA-directed silencing pathways in plants. Biochim Biophys Acta – Gene Regulatory Mechanisms 1779(11):720–724

    CAS  Google Scholar 

  19. Kurihara Y, Watanabe Y (2004) Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc Natl Acad Sci USA 101:12753–12758

    Article  CAS  PubMed  Google Scholar 

  20. Parizotto EA, Dunoyer P, Rahm N, Himber C, Voinnet O (2004) In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Genes Dev 18:2237–2242

    Article  CAS  PubMed  Google Scholar 

  21. Xie Z, Allen E, Fahlgren N, Calamar A, Givan SA, Carrington JC (2005) Expression of Arabidopsis MIRNA genes. Plant Physiol 138:2145–2154

    Article  CAS  PubMed  Google Scholar 

  22. Fang Y, Spector DL (2007) Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants. Curr Biol 17:818–823

    Article  CAS  PubMed  Google Scholar 

  23. Han MH, Goud S, Song L, Fedoroff N (2004) The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation. Proc Natl Acad Sci USA 101:1093–1098

    Article  CAS  PubMed  Google Scholar 

  24. Vazquez F, Gasciolli V, Crete P, Vaucheret H (2004) The nuclear dsRNA binding protein HYL1 is required for microRNA accumulation and plant development, but not posttranscriptional transgene silencing. Curr Biol 14:346–351

    CAS  PubMed  Google Scholar 

  25. Khvorova A, Reynolds A, Jayasena SD (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell 115:209–216

    Article  CAS  PubMed  Google Scholar 

  26. Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115:199–208

    Article  CAS  PubMed  Google Scholar 

  27. Baumberger N, Baulcombe DC (2005) Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci USA 102:11928–11933

    Article  CAS  PubMed  Google Scholar 

  28. Qi Y, Denli AM, Hannon GJ (2005) Biochemical specialization within Arabidopsis RNA silencing pathways. Mol Cell 19:421–428

    Article  CAS  PubMed  Google Scholar 

  29. Vaucheret H, Mallory AC, Bartel DP (2006) AGO1 homeostasis entails coexpression of MIR168 and AGO1 and preferential stabilization of miR168 by AGO1. Mol Cell 22:129–136

    Article  CAS  PubMed  Google Scholar 

  30. Hamilton A, Voinnet O, Chappell L, Baulcombe D (2002) Two classes of short interfering RNA in RNA silencing. Embo J 21:4671–4679

    Article  CAS  PubMed  Google Scholar 

  31. Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, Zilberman D, Jacobsen SE, Carrington JC (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2:E104

    Article  PubMed  Google Scholar 

  32. Herr AJ, Jensen MB, Dalmay T, Baulcombe DC (2005) RNA polymerase IV directs silencing of endogenous DNA. Science 308:118–120

    Article  CAS  PubMed  Google Scholar 

  33. Onodera Y, Haag JR, Ream T, Nunes PC, Pontes O, Pikaard CS (2005) Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell 120:613–622

    Article  CAS  PubMed  Google Scholar 

  34. Pontier D, Yahubyan G, Vega D, Bulski A, Saez-Vasquez J, Hakimi MA, Lerbs-Mache S, Colot V, Lagrange T (2005) Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis. Genes Dev 19:2030–2040

    Article  CAS  PubMed  Google Scholar 

  35. Zhang X, Henderson IR, Lu C, Green PJ, Jacobsen SE (2007) Role of RNA polymerase IV in plant small RNA metabolism. Proc Natl Acad Sci USA 104:4536–4541

    Article  CAS  PubMed  Google Scholar 

  36. Zilberman D, Cao X, Jacobsen SE (2003) ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 299:716–719

    Article  CAS  PubMed  Google Scholar 

  37. Li CF, Pontes O, El-Shami M, Henderson IR, Bernatavichute YV, Chan SW, Lagrange T, Pikaard CS, Jacobsen SE (2006) An ARGO­NAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126:93–106

    Article  CAS  PubMed  Google Scholar 

  38. Pontes O, Li CF, Nunes PC, Haag J, Ream T, Vitins A, Jacobsen SE, Pikaard CS (2006) The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell 126:79–92

    Article  CAS  PubMed  Google Scholar 

  39. Allen E, Xie Z, Gustafson AM, Carrington JC (2005) microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121:207–221

    Article  CAS  PubMed  Google Scholar 

  40. Axtell MJ, Jan C, Rajagopalan R, Bartel DP (2006) A two-hit trigger for siRNA biogenesis in plants. Cell 127:565–577

    Article  CAS  PubMed  Google Scholar 

  41. Peragine A, Yoshikawa M, Wu G, Albrecht HL, Poethig RS (2004) SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. Genes Dev 18:2368–2379

    Article  CAS  PubMed  Google Scholar 

  42. Vazquez F, Vaucheret H, Rajagopalan R, Lepers C, Gasciolli V, Mallory AC, Hilbert JL, Bartel DP, Crete P (2004) Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol Cell 16:69–79

    Article  CAS  PubMed  Google Scholar 

  43. Gasciolli V, Mallory AC, Bartel DP, Vaucheret H (2005) Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs. Curr Biol 15:1494–1500

    Article  CAS  PubMed  Google Scholar 

  44. Xie Z, Allen E, Wilken A, Carrington JC (2005) DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana. Proc Natl Acad Sci USA 102:12984–12989

    Article  CAS  PubMed  Google Scholar 

  45. Yoshikawa M, Peragine A, Park MY, Poethig RS (2005) A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev 19:2164–2175

    Article  CAS  PubMed  Google Scholar 

  46. Borsani O, Zhu J, Verslues PE, Sunkar R, Zhu JK (2005) Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell 123:1279–1291

    Article  CAS  PubMed  Google Scholar 

  47. Katiyar-Agarwal S, Morgan R, Dahlbeck D, Borsani O, Villegas A Jr, Zhu JK, Staskawicz BJ, Jin H (2006) A pathogen-inducible endogenous siRNA in plant immunity. Proc Natl Acad Sci USA 103:18002–18007

    Article  CAS  PubMed  Google Scholar 

  48. Yang Z, Ebright YW, Yu B, Chen X (2006) HEN1 recognizes 21–24 nt small RNA duplexes and deposits a methyl group onto the 2′ OH of the 3′ terminal nucleotide. Nucleic Acids Res 34:667–675

    Article  CAS  PubMed  Google Scholar 

  49. Yu B, Yang Z, Li J, Minakhina S, Yang M, Padgett RW, Steward R, Chen X (2005) Methylation as a crucial step in plant microRNA biogenesis. Science 307:932–935

    Article  CAS  PubMed  Google Scholar 

  50. Li J, Yang Z, Yu B, Liu J, Chen X (2005) Methylation protects miRNAs and siRNAs from a 3′-end uridylation activity in Arabidopsis. Curr Biol 15:1501–1507

    Article  CAS  PubMed  Google Scholar 

  51. Schauer SE, Jacobsen SE, Meinke DW, Ray A (2002) DICER-LIKE1: blind men and elephants in Arabidopsis development. Trends Plant Sci 7:487–491

    Article  CAS  PubMed  Google Scholar 

  52. Chen X, Liu J, Cheng Y, Jia D (2002) HEN1 functions pleiotropically in Arabidopsis development and acts in C function in the flower. Development 129:1085–1094

    Article  CAS  PubMed  Google Scholar 

  53. Golden TA, Schauer SE, Lang JD, Pien S, Mushegian AR, Grossniklaus U, Meinke DW, Ray A (2002) SHORT INTEGUMENTS1/SUSPENSOR1/CARPEL FACTORY, a Dicer homolog, is a maternal effect gene required for embryo development in Arabidopsis. Plant Physiol 130:808–822

    Article  PubMed  Google Scholar 

  54. Jacobsen SE, Running MP, Meyerowitz EM (1999) Disruption of an RNA helicase/RNAse III gene in Arabidopsis causes unregulated cell division in floral meristems. Development 126:5231–5243

    CAS  PubMed  Google Scholar 

  55. Ray A, Lang JD, Golden T, Ray S (1996) SHORT INTEGUMENTS1 (SIN1), a gene required for ovule development in Arabidopsis, also controls flowering time. Development 122:2631–2638

    CAS  PubMed  Google Scholar 

  56. Bohmert K, Camus I, Bellini C, Bouchez D, Caboche M, Benning C (1998) AGO1 defines a novel locus of Arabidopsis controlling leaf development. Embo J 17:170–180

    Article  CAS  PubMed  Google Scholar 

  57. Fagard M, Boutet S, Morel JB, Bellini C, Vaucheret H (2000) AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals. Proc Natl Acad Sci USA 97:11650–11654

    Article  CAS  PubMed  Google Scholar 

  58. Morel JB, Godon C, Mourrain P, Beclin C, Boutet S, Feuerbach F, Proux F, Vaucheret H (2002) Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance. Plant Cell 14:629–639

    Article  CAS  PubMed  Google Scholar 

  59. Kidner CA, Martienssen RA (2004) Spatially restricted microRNA directs leaf polarity through ARGONAUTE1. Nature 428:81–84

    Article  CAS  PubMed  Google Scholar 

  60. Lu C, Fedoroff N (2000) A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin. Plant Cell 12:2351–2366

    Article  CAS  PubMed  Google Scholar 

  61. Yang L, Liu Z, Lu F, Dong A, Huang H (2006) SERRATE is a novel nuclear regulator in primary microRNA processing in Arabidopsis. Plant J 47:841–850

    Article  CAS  PubMed  Google Scholar 

  62. Grigg SP, Canales C, Hay A, Tsiantis M (2005) SERRATE coordinates shoot meristem function and leaf axial patterning in Arabidopsis. Nature 437:1022–1026

    Article  CAS  PubMed  Google Scholar 

  63. Lobbes D, Rallapalli G, Schmidt DD, Martin C, Clarke J (2006) SERRATE: a new player on the plant microRNA scene. EMBO Rep 7:1052–1058

    Article  CAS  PubMed  Google Scholar 

  64. Kurihara Y, Takashi Y, Watanabe Y (2006) The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis. RNA 12:206–212

    Article  CAS  PubMed  Google Scholar 

  65. Adenot X, Elmayan T, Lauressergues D, Boutet S, Bouche N, Gasciolli V, Vaucheret H (2006) DRB4-dependent TAS3 trans-acting siRNAs control leaf morphology through AGO7. Curr Biol 16:927–932

    Article  CAS  PubMed  Google Scholar 

  66. Hiraguri A, Itoh R, Kondo N, Nomura Y, Aizawa D, Murai Y, Koiwa H, Seki M, Shinozaki K, Fukuhara T (2005) Specific interactions between Dicer-like proteins and HYL1/DRB-family dsRNA-binding proteins in Arabidopsis thaliana. Plant Mol Biol 57:173–188

    Article  CAS  PubMed  Google Scholar 

  67. Song L, Han MH, Lesicka J, Fedoroff N (2007) Arabidopsis primary microRNA processing proteins HYL1 and DCL1 define a nuclear body distinct from the Cajal body. Proc Natl Acad Sci USA 104:5437–5442

    Article  CAS  PubMed  Google Scholar 

  68. Telfer A, Poethig RS (1998) HASTY: a gene that regulates the timing of shoot maturation in Arabidopsis thaliana. Development 125:1889–1898

    CAS  PubMed  Google Scholar 

  69. Bollman KM, Aukerman MJ, Park MY, Hunter C, Berardini TZ, Poethig RS (2003) HASTY, the Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis. Development 130:1493–1504

    Article  CAS  PubMed  Google Scholar 

  70. Park MY, Wu G, Gonzalez-Sulser A, Vaucheret H, Poethig RS (2005) Nuclear processing and export of microRNAs in Arabidopsis. Proc Natl Acad Sci USA 102:3691–3696

    Article  CAS  PubMed  Google Scholar 

  71. Boutet S, Vazquez F, Liu J, Beclin C, Fagard M, Gratias A, Morel JB, Crete P, Chen X, Vaucheret H (2003) Arabidopsis HEN1: a genetic link between endogenous miRNA controlling development and siRNA controlling transgene silencing and virus resistance. Curr Biol 13:843–848

    Article  CAS  PubMed  Google Scholar 

  72. Stepanova AN, Alonso JM (2006) PCR-based screening for insertional mutants, in: Arabidopsis Protocols (Salinas J and Sanchez-Serrano J, eds) Humana Press, Totowa, NJ. pp. 163–172

    Google Scholar 

  73. Neff MM, Neff JD, Chory J, Pepper AE (1998) dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. Plant J 14:387–392

    Article  CAS  PubMed  Google Scholar 

  74. Valoczi A, Hornyik C, Varga N, Burgyan J, Kauppinen S, Havelda Z (2004) Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res 32:e175

    Article  PubMed  Google Scholar 

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Acknowledgments

I thank Chris Rock for critically reading the manuscript.

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Authors and Affiliations

  1. Department of Biological Sciences, Texas Tech University, Lubbock, TX, USA

    Zhixin Xie

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  1. Zhixin Xie
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Editors and Affiliations

  1. Delaware Biotechnology Institute, University of Delaware, Innovation Way 15, Newark, 19711, U.S.A.

    Blake C. Meyers

  2. Delaware Biotechnology Institute, University of Delaware, Innovation Way 15, Newark, 19711, U.S.A.

    Pamela J. Green

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Xie, Z. (2010). Piecing the Puzzle Together: Genetic Requirements for miRNA Biogenesis in Arabidopsis thaliana . In: Meyers, B., Green, P. (eds) Plant MicroRNAs. Methods in Molecular Biology, vol 592. Humana Press. https://doi.org/10.1007/978-1-60327-005-2_1

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  • DOI: https://doi.org/10.1007/978-1-60327-005-2_1

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  • Print ISBN: 978-1-60327-004-5

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