Analysis of siRNA Precursors Generated by RNA Polymerase IV and RNA-Dependent RNA Polymerase 2 in Arabidopsis

  • Todd BlevinsEmail author
  • Ram Podicheti
  • Craig S. Pikaard
Part of the Methods in Molecular Biology book series (MIMB, volume 1933)


Noncoding RNAs perform diverse regulatory functions in living cells. In plants, two RNA polymerase II-related enzymes, RNA polymerases IV and V (Pol IV and V), specialize in the synthesis of noncoding RNAs that silence a subset of transposable elements and genes via RNA-directed DNA methylation (RdDM). In this process, Pol IV partners with RNA-dependent RNA polymerase 2 (RDR2) to produce double-stranded RNAs that are then cut by an RNase III enzyme, Dicer-like 3 (DCL3), into 24 nt small interfering RNAs (siRNAs). The siRNAs are loaded into an Argonaute family protein, primarily AGO4, and guide the complex to complementary DNA target sequences where RdDM and repressive chromatin modifications ensue. The dependence of 24 nt siRNA biogenesis on Pol IV and RDR2 has been known for more than a decade, but the elusive pre-siRNA transcripts synthesized by Pol IV and RDR2 have only recently been identified. This chapter describes the approaches that enabled our identification of Pol IV/RDR2-dependent RNAs (P4R2 RNAs) in Arabidopsis thaliana. These included the use of a triple Dicer mutant (dcl2 dcl3 dcl4) to cause P4R2 RNAs to accumulate, genome-wide identification and mapping of P4R2 RNAs using a modified Illumina small RNA-Seq protocol, and multiple bioinformatic pipelines for data analysis and displaying results.

Key words

Nuclear multisubunit RNA polymerase IV (Pol IV) RNA-dependent RNA polymerase 2 (RDR2) RNA-directed DNA methylation (RdDM) siRNAs P4R2 RNAs 



The authors thank Doug Rusch and Haixu Tang for helping guide the informatics analyses, as well as Ross Cocklin for generating the rdr2 dcl2/3/4 quadruple mutant. C.S.P. is an Investigator at the Howard Hughes Medical Institute (HHMI) and former Plant Investigator of the Gordon and Betty Moore Foundation (GBMF). This work was supported by the National Institutes of Health (NIH) Grant GM077590, GBMF Grant GBMF3036 (to C.S.P.), and Investigator support funding of C.S.P. from HHMI. T.B. was supported, in part, by an NIH Ruth L. Kirschstein National Research Service Award. T.B. is currently supported by the LabEx consortium ANR-10-LABX-0036_NETRNA (“Investissements d’Avenir”) and by the French Agence Nationale de la Recherche (ANR) Grant ANR-17-CE20-0004-01.


  1. 1.
    Wilson RC, Doudna JA (2013) Molecular mechanisms of RNA interference. Annu Rev Biophys 42:217–239. Scholar
  2. 2.
    Vaucheret H (2008) Plant ARGONAUTES. Trends Plant Sci 13(7):350–358. Scholar
  3. 3.
    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(5):E104. Scholar
  4. 4.
    Yoshikawa M, Peragine A, Park MY, Poethig RS (2005) A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev 19(18):2164–2175. Scholar
  5. 5.
    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(1):69–79CrossRefGoogle Scholar
  6. 6.
    Llave C, Xie Z, Kasschau KD, Carrington JC (2002) Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297(5589):2053–2056. Scholar
  7. 7.
    Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M, Dunoyer P, Yamamoto YY, Sieburth L, Voinnet O (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320(5880):1185–1190. Scholar
  8. 8.
    Matzke MA, Kanno T, Matzke AJ (2015) RNA-Directed DNA methylation: the evolution of a complex epigenetic pathway in flowering plants. Annu Rev Plant Biol 66:243–267. Scholar
  9. 9.
    Ream TS, Haag JR, Wierzbicki AT, Nicora CD, Norbeck AD, Zhu JK, Hagen G, Guilfoyle TJ, Pasa-Tolic L, Pikaard CS (2009) Subunit compositions of the RNA-silencing enzymes Pol IV and Pol V reveal their origins as specialized forms of RNA polymerase II. Mol Cell 33(2):192–203. Scholar
  10. 10.
    Wendte JM, Pikaard CS (2017) The RNAs of RNA-directed DNA methylation. Biochim Biophys Acta 1860(1):140–148. Scholar
  11. 11.
    Law JA, Du J, Hale CJ, Feng S, Krajewski K, Palanca AM, Strahl BD, Patel DJ, Jacobsen SE (2013) Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature 498(7454):385–389. Scholar
  12. 12.
    Blevins T, Pontvianne F, Cocklin R, Podicheti R, Chandrasekhara C, Yerneni S, Braun C, Lee B, Rusch D, Mockaitis K, Tang H, Pikaard CS (2014) A two-step process for epigenetic inheritance in Arabidopsis. Mol Cell 54(1):30–42. Scholar
  13. 13.
    Haag JR, Ream TS, Marasco M, Nicora CD, Norbeck AD, Pasa-Tolic L, Pikaard CS (2012) In vitro transcription activities of Pol IV, Pol V, and RDR2 reveal coupling of Pol IV and RDR2 for dsRNA synthesis in plant RNA silencing. Mol Cell 48(5):811–818. Scholar
  14. 14.
    Wierzbicki AT, Ream TS, Haag JR, Pikaard CS (2009) RNA polymerase V transcription guides ARGONAUTE4 to chromatin. Nat Genet 41(5):630–634. Scholar
  15. 15.
    Lahmy S, Pontier D, Bies-Etheve N, Laudie M, Feng S, Jobet E, Hale CJ, Cooke R, Hakimi MA, Angelov D, Jacobsen SE, Lagrange T (2016) Evidence for ARGONAUTE4-DNA interactions in RNA-directed DNA methylation in plants. Genes Dev 30(23):2565–2570. Scholar
  16. 16.
    Zhong X, Du J, Hale CJ, Gallego-Bartolome J, Feng S, Vashisht AA, Chory J, Wohlschlegel JA, Patel DJ, Jacobsen SE (2014) Molecular mechanism of action of plant DRM de novo DNA methyltransferases. Cell 157(5):1050–1060. Scholar
  17. 17.
    Blevins T, Pontes O, Pikaard CS, Meins F Jr (2009) Heterochromatic siRNAs and DDM1 independently silence aberrant 5S rDNA transcripts in Arabidopsis. PLoS One 4(6):e5932. Scholar
  18. 18.
    Blevins T, Podicheti R, Mishra V, Marasco M, Wang J, Rusch D, Tang H, Pikaard CS (2015) Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis. elife 4:e09591. Scholar
  19. 19.
    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(1):79–92CrossRefGoogle Scholar
  20. 20.
    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(6):721–725 Epub 2006 May 2014CrossRefGoogle Scholar
  21. 21.
    Blevins T, Rajeswaran R, Aregger M, Borah BK, Schepetilnikov M, Baerlocher L, Farinelli L, Meins F Jr, Hohn T, Pooggin MM (2011) Massive production of small RNAs from a non-coding region of Cauliflower mosaic virus in plant defense and viral counter-defense. Nucleic Acids Res 39(12):5003–5014. Scholar
  22. 22.
    Axtell MJ (2013) Classification and comparison of small RNAs from plants. Annu Rev Plant Biol 64:137–159. Scholar
  23. 23.
    Rajagopalan R, Vaucheret H, Trejo J, Bartel DP (2006) A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev 20(24):3407–3425CrossRefGoogle Scholar
  24. 24.
    Kasschau KD, Fahlgren N, Chapman EJ, Sullivan CM, Cumbie JS, Givan SA, Carrington JC (2007) Genome-wide profiling and analysis of Arabidopsis siRNAs. PLoS Biol 5(3):e57CrossRefGoogle Scholar
  25. 25.
    Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15(2):188–200CrossRefGoogle Scholar
  26. 26.
    Lu C, Meyers BC, Green PJ (2007) Construction of small RNA cDNA libraries for deep sequencing. Methods 43(2):110–117. Scholar
  27. 27.
    Zhai J, Bischof S, Wang H, Feng S, Lee TF, Teng C, Chen X, Park SY, Liu L, Gallego-Bartolome J, Liu W, Henderson IR, Meyers BC, Ausin I, Jacobsen SE (2015) A one precursor one siRNA model for Pol IV-dependent siRNA biogenesis. Cell 163(2):445–455. Scholar
  28. 28.
    Coruh C, Cho SH, Shahid S, Liu Q, Wierzbicki A, Axtell MJ (2015) Comprehensive annotation of Physcomitrella patens small RNA loci reveals that the heterochromatic short interfering RNA pathway is largely conserved in land plants. Plant Cell 27(8):2148–2162. Scholar
  29. 29.
    Fei Q, Xia R, Meyers BC (2013) Phased, secondary, small interfering RNAs in posttranscriptional regulatory networks. Plant Cell 25(7):2400–2415. Scholar
  30. 30.
    Onodera Y, Haag JR, Ream T, Costa Nunes P, Pontes O, Pikaard CS (2005) Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell 120(5):613–622. Scholar
  31. 31.
    Blevins T, Rajeswaran R, Shivaprasad PV, Beknazariants D, Si-Ammour A, Park HS, Vazquez F, Robertson D, Meins F Jr, Hohn T, Pooggin MM (2006) Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing. Nucleic Acids Res 34(21):6233–6246. Scholar
  32. 32.
    Blevins T (2017) Northern blotting techniques for small RNAs. Methods Mol Biol 1456:141–162. Scholar
  33. 33.
    Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15):2114–2120. Scholar
  34. 34.
    Chandrasekhara C, Mohannath G, Blevins T, Pontvianne F, Pikaard CS (2016) Chromosome-specific NOR inactivation explains selective rRNA gene silencing and dosage control in Arabidopsis. Genes Dev 30(2):177–190. Scholar
  35. 35.
    Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3):R25. Scholar
  36. 36.
    Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25(16):2078–2079. Scholar
  37. 37.
    Skinner ME, Uzilov AV, Stein LD, Mungall CJ, Holmes IH (2009) JBrowse: a next-generation genome browser. Genome Res 19(9):1630–1638. Scholar
  38. 38.
    Podicheti R, Mockaitis K (2015) FEATnotator: a tool for integrated annotation of sequence features and variation, facilitating interpretation in genomics experiments. Methods 79–80:11–17. Scholar
  39. 39.
  40. 40.
    Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: a sequence logo generator. Genome Res 14(6):1188–1190. Scholar
  41. 41.
    Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, Mitros T, Dirks W, Hellsten U, Putnam N, Rokhsar DS (2012) Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res 40(Database issue):D1178–D1186. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Todd Blevins
    • 1
    • 2
    • 3
    • 4
    Email author
  • Ram Podicheti
    • 5
    • 6
  • Craig S. Pikaard
    • 1
    • 2
    • 3
  1. 1.Howard Hughes Medical InstituteIndiana UniversityBloomingtonUSA
  2. 2.Department of BiologyIndiana UniversityBloomingtonUSA
  3. 3.Department of Molecular and Cellular BiochemistryIndiana UniversityBloomingtonUSA
  4. 4.Institut de Biologie Moléculaire des Plantes, CNRS UPR2357Université de StrasbourgStrasbourgFrance
  5. 5.Center for Genomics and BioinformaticsIndiana UniversityBloomingtonUSA
  6. 6.School of Informatics, Computing and EngineeringIndiana UniversityBloomingtonUSA

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