Analysis of the Uridylation of Both ARGONAUTE-Bound MiRNAs and 5′ Cleavage Products of Their Target RNAs in Plants

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1640)

Abstract

Uridylation (3′ untemplated uridine addition) provides a mechanism to trigger the degradation of miRNAs and the 5′ cleavage products (5′ CP) that are produced from miRNA-directed ARGONAUTE (AGO) cleavage of target RNAs. We have recently shown that HEN1 SUPPRESSOR 1 (HESO1), a terminal uridylyltransferase, and its homolog UTP:RNA uridylyltransferase 1 (URT1) catalyze the uridylation of miRNAs and 5′ CPs within the AGO complex in higher plants. In this chapter, we describe detailed protocols for analyzing 3′ end uridylation of both AGO-bound miRNAs and 5′ CP.

Key words

miRNA ARGONAUTE 5′ Cleavage product Uridylation HESO1 Rapid amplification of cDNA ends (RACE) 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

We thank Yuan Wang for helping with the preparation of the figure. This work was supported by the Ministry of Science and Technology of China (Grant No. 2016YFA0503200) and National Natural Science Foundation of China (Grant Nos. 31471221 and 31622009) to G.R. and Nebraska Soybean Board (Grant No. 16R–05–3/3 #1706) and National Science Foundation (Award No. OIA–1557417) to B.Y.

No conflict of interest was declared.

References

  1. 1.
    Chen X (2009) Small RNAs and their roles in plant development. Annu Rev Cell Dev Biol 25:21–44. doi: 10.1146/annurev.cellbio.042308.113417 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215–233. doi: 10.1016/j.cell.2009.01.002 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Ren G, Yu B (2012) Critical roles of RNA-binding proteins in miRNA biogenesis in Arabidopsis. RNA Biol 9(12):1424–1428. doi: 10.4161/rna.22740 CrossRefPubMedGoogle Scholar
  4. 4.
    Ha M, Kim VN (2014) Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 15(8):509–524. doi: 10.1038/nrm3838 CrossRefPubMedGoogle Scholar
  5. 5.
    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(5711):932–935. doi: 10.1126/science.1107130 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP (2002) Prediction of plant microRNA targets. Cell 110(4):513–520. doi: 10.1016/S0092-8674(02)00863-2 CrossRefPubMedGoogle Scholar
  7. 7.
    Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB (2003) Prediction of mammalian microRNA targets. Cell 115(7):787–798. doi: 10.1016/S0092-8674(03)01018-3 CrossRefPubMedGoogle Scholar
  8. 8.
    Meister G (2013) Argonaute proteins: functional insights and emerging roles. Nat Rev Genet 14(7):447–459. doi: 10.1038/nrg3462 CrossRefPubMedGoogle Scholar
  9. 9.
    Schwab R, Palatnik JF, Riester M, Schommer C, Schmid M, Weigel D (2005) Specific effects of microRNAs on the plant transcriptome. Dev Cell 8(4):517–527. doi: 10.1016/j.devcel.2005.01.018 CrossRefPubMedGoogle Scholar
  10. 10.
    Yekta S, Shih IH, Bartel DP (2004) MicroRNA-directed cleavage of HOXB8 mRNA. Science 304(5670):594–596. doi: 10.1126/science.1097434 CrossRefPubMedGoogle Scholar
  11. 11.
    Shin C, Nam JW, Farh KK, Chiang HR, Shkumatava A, Bartel DP (2010) Expanding the microRNA targeting code: functional sites with centered pairing. Mol Cell 38(6):789–802. doi: 10.1016/j.molcel.2010.06.005 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Zhang X, Zhu Y, Liu X, Hong X, Xu Y, Zhu P, Shen Y, Wu H, Ji Y, Wen X, Zhang C, Zhao Q, Wang Y, Lu J, Guo H (2015) Plant biology. Suppression of endogenous gene silencing by bidirectional cytoplasmic RNA decay in Arabidopsis. Science 348(6230):120–123. doi: 10.1126/science.aaa2618 CrossRefPubMedGoogle Scholar
  13. 13.
    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(16):1501–1507. doi: 10.1016/j.cub.2005.07.029 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Lee M, Kim B, Kim VN (2014) Emerging roles of RNA modification: m(6)A and U-tail. Cell 158(5):980–987. doi: 10.1016/j.cell.2014.08.005 CrossRefPubMedGoogle Scholar
  15. 15.
    Ren G, Chen X, Yu B (2014) Small RNAs meet their targets: when methylation defends miRNAs from uridylation. RNA Biol 11(9):1099–1104. doi: 10.4161/rna.36243 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Ren G, Xie M, Zhang S, Vinovskis C, Chen X, Yu B (2014) Methylation protects microRNAs from an AGO1-associated activity that uridylates 5′ RNA fragments generated by AGO1 cleavage. Proc Natl Acad Sci U S A 111(17):6365–6370. doi: 10.1073/pnas.1405083111 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Shen B, Goodman HM (2004) Uridine addition after microRNA-directed cleavage. Science 306(5698):997. doi: 10.1126/science.1103521 CrossRefPubMedGoogle Scholar
  18. 18.
    Tu B, Liu L, Xu C, Zhai J, Li S, Lopez MA, Zhao Y, Yu Y, Ramachandran V, Ren G, Yu B, Li S, Meyers BC, Mo B, Chen X (2015) Distinct and cooperative activities of HESO1 and URT1 nucleotidyl transferases in microRNA turnover in Arabidopsis. PLoS Genet 11(4):e1005119. doi: 10.1371/journal.pgen.1005119 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Wang X, Zhang S, Dou Y, Zhang C, Chen X, Yu B, Ren G (2015) Synergistic and independent actions of multiple terminal nucleotidyl transferases in the 3′ tailing of small RNAs in Arabidopsis. PLoS Genet 11(4):e1005091. doi: 10.1371/journal.pgen.1005091 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Ren G, Chen X, Yu B (2012) Uridylation of miRNAs by hen1 suppressor1 in Arabidopsis. Curr Biol 22(8):695–700. doi: 10.1016/j.cub.2012.02.052 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Zhao Y, Yu Y, Zhai J, Ramachandran V, Dinh TT, Meyers BC, Mo B, Chen X (2012) The Arabidopsis nucleotidyl transferase HESO1 uridylates unmethylated small RNAs to trigger their degradation. Curr Biol 22(8):689–694. doi: 10.1016/j.cub.2012.02.051 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33(5):949–956. doi: 10.1046/j.1365-313X.2003.01676.x CrossRefPubMedGoogle Scholar
  23. 23.
    Qi Y, Denli AM, Hannon GJ (2005) Biochemical specialization within Arabidopsis RNA silencing pathways. Mol Cell 19(3):421–428. doi: 10.1016/j.molcel.2005.06.014 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.State Key Laboratory of Genetic Engineering and Collaborative Innovation Center for Genetics and DevelopmentSchool of Life Sciences, Fudan UniversityShanghaiChina
  2. 2.Center for Plant Science Innovation and School of Biological SciencesUniversity of Nebraska–LincolnLincolnUSA

Personalised recommendations