Abstract
MicroRNAs (miRNAs) originate from primary transcripts (pri-miRNAs) with characteristic stem-loop structures, and their accurate processing is required for the production of functional miRNAs. Here, using the pri-miR-166 family in Arabidopsis thaliana as a paradigm, we report the crucial role of pri-miRNA terminal loops in miRNA biogenesis. We found that multibranched terminal loops in pri-miR-166s substantially suppress miR-166 expression in vivo. Unlike canonical processing of pri-miRNAs, terminal loop–branched pri-miRNAs can be processed by Dicer-like 1 (DCL1) complexes bidirectionally from base to loop and from loop to base, resulting in productive and abortive processing of miRNAs, respectively. In both cases, DCL1 complexes canonically cut pri-miRNAs at a distance of 16–17 bp from a reference single-stranded loop region. DCL1 also adjusts processing sites toward an internal loop through its helicase domain. These results provide new insight into the poorly understood processing mechanism of pri-miRNAs with complex secondary structures.
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References
Kim, V.N., Han, J. & Siomi, M.C. Biogenesis of small RNAs in animals. Nat. Rev. Mol. Cell Biol. 10, 126–139 (2009).
Han, J. et al. Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell 125, 887–901 (2006).
Bartel, D.P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).
Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004).
Voinnet, O. Origin, biogenesis and activity of plant microRNAs. Cell 136, 669–687 (2009).
Guo, H., Ingolia, N.T., Weissman, J.S. & Bartel, D.P. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835–840 (2010).
Czech, B. & Hannon, G.J. Small RNA sorting: matchmaking for Argonautes. Nat. Rev. Genet. 12, 19–31 (2011).
Bazzini, A.A., Lee, M. & Giraldez, A. Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish. Science 336, 233–237 (2012).
Djuranovic, S., Nahvi, A. & Green, R. miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay. Science 336, 237–240 (2012).
Li, S. et al. MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis. Cell 153, 562–574 (2013).
Chen, X. Plant microRNAs at a glance. Semin. Cell Dev. Biol. 21, 781 (2010).
Ren, G. et al. Regulation of miRNA abundance by RNA binding protein TOUGH in Arabidopsis. Proc. Natl. Acad. Sci. USA 109, 12817–12821 (2012).
Machida, S., Chen, H. & Yuan, A. Molecular insights into miRNA processing by Arabidopsis thaliana SERRATE. Nucleic Acids Res. 39, 7828–7836 (2011).
Yang, S.W. et al. Structure of Arabidopsis HYPONASTIC LEAVES1 and its molecular implications for miRNA processing. Structure 18, 594–605 (2010).
Dong, Z., Han, M. & Fedoroff, N. The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1. Proc. Natl. Acad. Sci. USA 105, 9970–9975 (2008).
Grigg, S.P., Canales, C., Hay, A. & Tsiantis, M. SERRATE coordinates shoot meristem function and leaf axial patterning in Arabidopsis. Nature 437, 1022–1026 (2005).
Han, M.H., Goud, S., Song, L. & Fedoroff, N. The Arabidopsis double-stranded RNA binding protein HYL1 plays a role in microRNA-mediated gene regulation. Proc. Natl. Acad. Sci. USA 101, 1093–1098 (2004).
Kurihara, Y., Takashi, Y. & Watanabe, Y. The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis. RNA 12, 206–212 (2006).
Lobbes, D., Rallapalli, G., Schmidt, D., Martin, C. & Clarke, J. SERRATE: a new player on the plant microRNA scene. EMBO Rep. 7, 1052–1058 (2006).
Vazquez, F., Gasciolli, V., Crete, P. & Vaucheret, H. The nuclear dsRNA binding protein HYL1 is required for microRNA accumulation and plant development, but not posttranscriptional transgene silencing. Curr. Biol. 14, 346–351 (2004).
Hirsch, J. et al. Characterization of 43 non–protein-coding mRNA genes in Arabidopsis, including the MIR162a-derived transcripts. Plant Physiol. 140, 1192–1204 (2006).
Xie, Z. et al. Expression of Arabidopsis MIRNA genes. Plant Physiol. 138, 2145–2154 (2005).
Cuperus, J.T. et al. Identification of MIR390a precursor processing-defective mutants in Arabidopsis by direct genome sequencing. Proc. Natl. Acad. Sci. USA 107, 466–471 (2010).
Mateos, J.L., Bologna, N.G., Chorostecki, U. & Palatnik, J.F. Identification of microRNA processing determinants by random mutagenesis of Arabidopsis MIR172a precursor. Curr. Biol. 20, 49–54 (2010).
Song, L., Axtell, M. & Fedoroff, N. RNA secondary structural determinants of miRNA precursor processing in Arabidopsis. Curr. Biol. 20, 37–41 (2010).
Werner, S., Wollmann, H., Schneeberger, K. & Weigel, D. Structure determinants for accurate processing of miR172a in Arabidopsis thaliana. Curr. Biol. 20, 42–48 (2010).
Addo-Quaye, C. et al. Sliced microRNA targets and precise loop-first processing of MIR319 hairpins revealed by analysis of the Physcomitrella patens degradome. RNA 15, 2112–2121 (2009).
Bologna, N.G., Mateos, J.L., Bresso, E.G. & Palatnik, J.F. A loop to base processing mechanism underlies the biogenesis of plant microRNAs miR319 and miR159. EMBO J. 28, 3646–3656 (2009).
Zhu, H. et al. Arabidopsis Argonaute10 specifically sequesters miR166/165 to regulate shoot apical meristem development. Cell 145, 242–256 (2011).
Zhang, Z. & Zhang, X. Argonautes compete for miR165/166 to regulate shoot apical meristem development. Curr. Opin. Plant Biol. 15, 652–658 (2012).
Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).
Liu, C., Axtell, M.J. & Fedoroff, N.V. The helicase and RNase IIIa domains of Arabidopsis Dicer-like 1 modulate catalytic parameters during microRNA biogenesis. Plant Physiol. 159, 748–758 (2012).
Manavella, P.A. et al. Fast-forward genetics identifies plant CPL phosphatases as regulators of miRNA processing factor HYL1. Cell 151, 859–870 (2012).
Weinberg, D.E., Kotaro, N.K., Dinshaw J. Patel, D. & Bartel, D. The inside-out mechanism of Dicers from budding yeasts. Cell 146, 262–276 (2011).
Gu, S. et al. The loop position of shRNAs and pre-miRNAs is critical for the accuracy of Dicer processing in vivo. Cell 151, 900–911 (2012).
Tsutsumi, A., Kawamata, T., Izumi, N., Seitz, H. & Tomari, Y. Recognition of the pre-miRNA structure by Drosophila Dicer-1. Nat. Struct. Mol. Biol. 18, 1153–1158 (2011).
Addo-Quaye, C., Eshoo, T.W., Bartel, D.P. & Axtell, M.J. Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome. Curr. Biol. 18, 758–762 (2008).
German, M.A. et al. Global identification of microRNA-target RNA pairs by parallel analysis of RNA ends. Nat. Biotechnol. 26, 941–946 (2008).
Gregory, B.D. et al. A link between RNA metabolism and silencing affecting Arabidopsis development. Dev. Cell 14, 854–866 (2008).
Ma, Z., Coruh, C. & Axtell, M.J. Arabidopsis lyrata small RNAs: transient miRNA and small interfering RNA loci within the Arabidopsis genus. Plant Cell 22, 1090–1103 (2010).
Barrett, T. et al. NCBI GEO: archive for functional genomics data sets—update. Nucleic Acids Res. 41, D991–D995 (2013).
Zhang, X., Henriques, R., Lin, S.S., Niu, Q. & Chua, N.H. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral-dip method. Nat. Protoc. 1, 641–646 (2006).
Zhang, X. et al. Cucumber mosaic virus–encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense. Genes Dev. 20, 3255–3268 (2006).
Zhang, X., Garreton, V. & Chua, N.H. The AIP2 E3 ligase acts as a novel negative regulator of ABA signaling by promoting ABI3 degradation. Genes Dev. 19, 1532–1543 (2005).
Acknowledgements
We thank H. Koiwa for pDONRzeo-HYL1 (Department of Horticulture, Texas A&M University) and D. Shippen, G. Kapler, F. Qiao, P.W. Li and M. Klein for stimulating discussions and critical review of the manuscript. We also thank F. Hu, H. Xu, Z. Zhang, C. Huang and C. Lu for technical assistance. The work was supported by grants from the US National Science Foundation (NSF) (MCB-0951120) and NSF CAREER (MCB-1253369), the US National Institutes of Health (R21AI097570) and the Welch foundation (A-1777) to X.Z. A.L. was supported by NSF-REU (MCB-1232817). Y.Z. was supported by the Chinese Scholarship Council.
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H.Z., C.C.-G. and X.Z. designed experiments. Y.Z. carried out genetic research. H.Z. and C.C.-G. performed biochemical studies. M.J.A. conducted degradome analysis. Y.-T.Z. and X.-J.W. worked on sRNA data set analysis. A.L., C.G., L.D. and Z.L. participated in experiments or provided materials and intellectual input for the work. X.Z. and H.Z. wrote the manuscript.
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Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–9 (PDF 8269 kb)
Supplementary Table 1
Primers used in this study. (XLSX 28 kb)
Supplementary Table 2
Reads of sRNAs generated from bi-directional processing of selected Arabidopsis pri-miRNAs. (XLSX 26 kb)
Supplementary Table 3
Reads of sRNAs generated from bi-directional processing of selected primiRNAs in rice. (XLSX 19 kb)
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Zhu, H., Zhou, Y., Castillo-González, C. et al. Bidirectional processing of pri-miRNAs with branched terminal loops by Arabidopsis Dicer-like1. Nat Struct Mol Biol 20, 1106–1115 (2013). https://doi.org/10.1038/nsmb.2646
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DOI: https://doi.org/10.1038/nsmb.2646
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