Advertisement

Detection of MicroRNA Processing Intermediates Through RNA Ligation Approaches

  • Belén MoroEmail author
  • Arantxa M. L. Rojas
  • Javier F. PalatnikEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1932)

Abstract

MicroRNAs (miRNA) are small RNAs of 20–22 nt that regulate diverse biological pathways through the modulation of gene expression. miRNAs recognize target RNAs by base complementarity and guide them to degradation or translational arrest. They are transcribed as longer precursors with extensive secondary structures. In plants, these precursors are processed by a complex harboring DICER-LIKE1 (DCL1), which cuts on the precursor stem region to release the mature miRNA together with the miRNA*. In both plants and animals, the miRNA precursors contain spatial clues that determine the position of the miRNA along their sequences. DCL1 is assisted by several proteins, such as the double-stranded RNA binding protein, HYPONASTIC LEAVES1 (HYL1), and the zinc finger protein SERRATE (SE). The precise biogenesis of miRNAs is of utter importance since it determines the exact nucleotide sequence of the mature small RNAs and therefore the identity of the target genes. miRNA processing itself can be regulated and therefore can determine the final small RNA levels and activity. Here, we describe methods to analyze miRNA processing intermediates in plants. These approaches can be used in wild-type or mutant plants, as well as in plants grown under different conditions, allowing a molecular characterization of the miRNA biogenesis from the RNA precursor perspective.

Key words

MicroRNA Processing Precursor RNA ligation NGS Plants Arabidopsis 

Notes

Acknowledgments

Supported by grants of Argentinean Ministry of Science, PICT-2015-3557 and PICT-2016-0761 to J.P. CONICET fellowships to B.M. and A.M.L.R.

References

  1. 1.
    Bologna NG, Voinnet O (2014) The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu Rev Plant Biol 65:473–503CrossRefGoogle Scholar
  2. 2.
    Ha M, Kim VN (2014) Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 15:509–524CrossRefGoogle Scholar
  3. 3.
    Axtell MJ (2008) Evolution of microRNAs and their targets: are all microRNAs biologically relevant? Biochim Biophys Acta 1779:725–734CrossRefGoogle Scholar
  4. 4.
    Cuperus JT, Fahlgren N, Carrington JC (2011) Evolution and functional diversification of MIRNA genes. Plant Cell 23:431–442CrossRefGoogle Scholar
  5. 5.
    Voinnet O (2009) Origin, biogenesis, and activity of plant microRNAs. Cell 136:669–687CrossRefGoogle Scholar
  6. 6.
    Rodriguez RE, Schommer C, Palatnik JF (2016) Control of cell proliferation by microRNAs in plants. Curr Opin Plant Biol 34:68–76CrossRefGoogle Scholar
  7. 7.
    Rogers K, Chen X (2013) Biogenesis, turnover, and mode of action of plant microRNAs. Plant Cell 25:2383–2399CrossRefGoogle Scholar
  8. 8.
    Xie Z, Allen E, Fahlgren N, Calamar A, Givan SA, Carrington JC (2005) Expression of Arabidopsis MIRNA genes. Plant Physiol 138:2145–2154CrossRefGoogle Scholar
  9. 9.
    Meyers BC, Axtell MJ, Bartel B, Bartel DP, Baulcombe D, Bowman JL, Cao X, Carrington JC, Chen X, Green PJ, Griffiths-Jones S, Jacobsen SE, Mallory AC, Martienssen RA, Poethig RS, Qi Y, Vaucheret H, Voinnet O, Watanabe Y, Weigel D, Zhu JK (2008) Criteria for annotation of plant microRNAs. Plant Cell 20:3186–3190CrossRefGoogle Scholar
  10. 10.
    Lee Y, Jeon K, Lee JT, Kim S, Kim VN (2002) MicroRNA maturation: stepwise processing and subcellular localization. EMBO J 21:4663–4670CrossRefGoogle Scholar
  11. 11.
    Han J, Lee Y, Yeom KH, Kim YK, Jin H, Kim VN (2004) The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev 18:3016–3027CrossRefGoogle Scholar
  12. 12.
    Auyeung Vincent C, Ulitsky I, McGeary Sean E, Bartel David P (2013) Beyond secondary structure: primary-sequence determinants license pri-miRNA hairpins for processing. Cell 152:844–858CrossRefGoogle Scholar
  13. 13.
    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 U S A 101:1093–1098CrossRefGoogle Scholar
  14. 14.
    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 14:346–351CrossRefGoogle Scholar
  15. 15.
    Lobbes D, Rallapalli G, Schmidt DD, Martin C, Clarke J (2006) SERRATE: a new player on the plant microRNA scene. EMBO Rep 7:1052–1058CrossRefGoogle Scholar
  16. 16.
    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 47:841–850CrossRefGoogle Scholar
  17. 17.
    Gregory BD, O’Malley RC, Lister R, Urich MA, Tonti-Filippini J, Chen H, Millar AH, Ecker JR (2008) A link between RNA metabolism and silencing affecting Arabidopsis development. Dev Cell 14:854–866CrossRefGoogle Scholar
  18. 18.
    Laubinger S, Sachsenberg T, Zeller G, Busch W, Lohmann JU, Ratsch G, Weigel D (2008) Dual roles of the nuclear cap-binding complex and SERRATE in pre-mRNA splicing and microRNA processing in Arabidopsis thaliana. Proc Natl Acad Sci U S A 105:8795–8800CrossRefGoogle Scholar
  19. 19.
    Kim S, Yang J-Y, Xu J, Jang I-C, Prigge MJ, Chua N-H (2008) Two cap-binding proteins CBP20 and CBP80 are involved in processing primary MicroRNAs. Plant Cell Physiol 49:1634–1644CrossRefGoogle Scholar
  20. 20.
    Ren G, Xie M, Dou Y, Zhang S, Zhang C, Yu B (2012) Regulation of miRNA abundance by RNA binding protein TOUGH in Arabidopsis. Proc Natl Acad Sci U S A 109:12817–12821CrossRefGoogle Scholar
  21. 21.
    Yu B, Bi L, Zheng B, Ji L, Chevalier D, Agarwal M, Ramachandran V, Li W, Lagrange T, Walker JC, Chen X (2008) The FHA domain proteins DAWDLE in Arabidopsis and SNIP1 in humans act in small RNA biogenesis. Proc Natl Acad Sci U S A 105:10073–10078CrossRefGoogle Scholar
  22. 22.
    Zhang Z, Guo X, Ge C, Ma Z, Jiang M, Li T, Koiwa H, Yang SW, Zhang X (2017) KETCH1 imports HYL1 to nucleus for miRNA biogenesis in Arabidopsis. Proc Natl Acad Sci U S A 114:4011–4016CrossRefGoogle Scholar
  23. 23.
    Manavella PA, Hagmann J, Ott F, Laubinger S, Franz M, Macek B, Weigel D (2012) Fast-forward genetics identifies plant CPL phosphatases as regulators of miRNA processing factor HYL1. Cell 151:859–870CrossRefGoogle Scholar
  24. 24.
    Axtell MJ, Westholm JO, Lai EC (2011) Vive la difference: biogenesis and evolution of microRNAs in plants and animals. Genome Biol 12:221CrossRefGoogle Scholar
  25. 25.
    Han J, Lee Y, Yeom KH, Nam JW, Heo I, Rhee JK, Sohn SY, Cho Y, Zhang BT, Kim VN (2006) Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell 125:887–901CrossRefGoogle Scholar
  26. 26.
    Bologna NG, Mateos JL, Bresso EG, Palatnik JF (2009) A loop-to-base processing mechanism underlies the biogenesis of plant microRNAs miR319 and miR159. EMBO J 28:3646–3656CrossRefGoogle Scholar
  27. 27.
    Mateos JL, Bologna NG, Chorostecki U, Palatnik JF (2010) Identification of microRNA processing determinants by random mutagenesis of Arabidopsis MIR172a precursor. Curr Biol 20:49–54CrossRefGoogle Scholar
  28. 28.
    Song L, Axtell MJ, Fedoroff NV (2010) RNA secondary structural determinants of miRNA precursor processing in Arabidopsis. Curr Biol 20:37–41CrossRefGoogle Scholar
  29. 29.
    Werner S, Wollmann H, Schneeberger K, Weigel D (2010) Structure determinants for accurate processing of miR172a in Arabidopsis thaliana. Curr Biol 20:42–48CrossRefGoogle Scholar
  30. 30.
    Bologna NG, Schapire AL, Zhai J, Chorostecki U, Boisbouvier J, Meyers BC, Palatnik JF (2013) Multiple RNA recognition patterns during microRNA biogenesis in plants. Genome Res 23:1675–1689CrossRefGoogle Scholar
  31. 31.
    Chorostecki U, Moro B, Rojas ALM, Debernardi JM, Schapire AL, Notredame C, Palatnik J (2017) Evolutionary footprints reveal insights into plant microRNA biogenesis. Plant Cell 29:1248–1261PubMedPubMedCentralGoogle Scholar
  32. 32.
    Kim W, Kim H-E, Jun AR, Jung MG, Jin S, Lee J-H, Ahn JH (2016) Structural determinants of miR156a precursor processing in temperature-responsive flowering in Arabidopsis. J Exp Bot 67:4659–4670CrossRefGoogle Scholar
  33. 33.
    Kurihara Y, Watanabe Y (2004) Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc Natl Acad Sci U S A 101:12753–12758CrossRefGoogle Scholar
  34. 34.
    Addo-Quaye C, Snyder JA, Park YB, Li YF, Sunkar R, Axtell MJ (2009) Sliced microRNA targets and precise loop-first processing of MIR319 hairpins revealed by analysis of the Physcomitrella patens degradome. RNA 15:2112–2121CrossRefGoogle Scholar
  35. 35.
    Bologna NG, Schapire AL, Palatnik JF (2013) Processing of plant microRNA precursors. Brief Funct Genomics 12:37–45CrossRefGoogle Scholar
  36. 36.
    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 12:206–212CrossRefGoogle Scholar
  37. 37.
    Schapire AL, Bologna NG, Moro B, Zhai J, Meyers BC, Palatnik JF (2013) Construction of Specific Parallel Amplification of RNA Ends (SPARE) libraries for the systematic identification of plant microRNA processing intermediates. Methods 64:283–291CrossRefGoogle Scholar
  38. 38.
    Moro B, Chorostecki U, Arikit S, Suarez IP, Höbartner C, Rasia RM, Meyers BC, Palatnik JF (2018) Efficiency and precision of microRNA biogenesis modes in plants. Nucleic acids research 46(20):10709–10723.  https://doi.org/10.1093/nar/gky853
  39. 39.
    Classic Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  40. 40.
    Hang R, Deng X, Liu C, Mo B, Cao X (2015) Circular RT-PCR assay using arabidopsis samples. Bio-protocol 5:e1533.  https://doi.org/10.21769/BioProtoc.1533CrossRefGoogle Scholar
  41. 41.
    Basyuk E, Suavet F, Doglio A, Bordonne R, Bertrand E (2003) Human let-7 stem-loop precursors harbor features of RNase III cleavage products. Nucleic Acids Res 31:6593–6597CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Instituto de Biología Molecular y Celular de Rosario, CONICETUniversidad Nacional de RosarioRosarioArgentina
  2. 2.Facultad de Ciencias Bioquímicas y FarmacéuticasUniversidad Nacional de RosarioRosarioArgentina
  3. 3.Centro de Estudios InterdisciplinariosUniversidad Nacional de RosarioRosarioArgentina

Personalised recommendations