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Bidirectional processing of pri-miRNAs with branched terminal loops by Arabidopsis Dicer-like1

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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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Figure 1: The secondary structures of pri-miRNAs affect miRNA abundance in vivo.
Figure 2: The concomitant presence of productive and abortive processing of pri-miR-166c but not pri-miR-166f.
Figure 3: DCL1 complexes process pri-miR-166c bidirectionally from lower stem to loop and from loop to lower stem.
Figure 4: DCL1 initiates cleavage at 16–17 bp from the ssRNA-dsRNA junction in pri-miRNAs.
Figure 5: DCL1 complexes adjust cleavage sites toward the edge of internal loops through the helicase domain.
Figure 6: Secondary structural elements are required for abortive processing in pri-miR-166c.
Figure 7: Bidirectional processing of pri-miRNAs (BTLs) extends beyond the pri-miR-166 family in Arabidopsis.
Figure 8: The terminal loop affects the steady-state abundance but not the processing pattern of pre-miRNAs.

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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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Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Xiuren Zhang.

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The authors declare no competing financial interests.

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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