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Unique functionality of 22-nt miRNAs in triggering RDR6-dependent siRNA biogenesis from target transcripts in Arabidopsis

Abstract

RNA interference pathways can involve amplification of secondary siRNAs by RNA-dependent RNA polymerases. In plants, RDR6-dependent secondary siRNAs arise from transcripts targeted by some microRNAs (miRNAs). Here, Arabidopsis thaliana secondary siRNAs from mRNA as well as trans-acting siRNAs are shown to be triggered through initial targeting by a 22-nucleotide (nt) miRNA that associates with AGO1. In contrast to canonical 21-nt miRNAs, 22-nt miRNAs primarily arise from foldback precursors containing asymmetric bulges. Using artificial miRNA constructs, conversion of asymmetric foldbacks to symmetric foldbacks resulted in the production of 21-nt forms of miR173, miR472 and miR828. Both 21- and 22-nt forms associated with AGO1 and guided accurate slicer activity, but only 22-nt forms were competent to trigger RDR6-dependent siRNA production from target RNA. These data suggest that AGO1 functions differentially with 21- and 22-nt miRNAs to engage the RDR6-associated amplification apparatus.

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Figure 1: Small RNAs from Arabidopsis annotated transcripts.
Figure 2: MIRNA foldback asymmetry leads to the formation of 22-nt miRNAs.
Figure 3: Production and activities of 21- and 22-nt miR173 forms.
Figure 4: Production and activities of 21- and 22-nt miR472 and miR828 forms.

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Acknowledgements

A.C. was supported by a postdoctoral fellowship from the Spanish Ministerio de Ciencia e Innovacion (BMC-2008-0188). This work was supported by grants from the US National Science Foundation (MCB-0618433 and MCB-0956526), the US National Institutes of Health (AI43288) and Monsanto Corporation.

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J.T.C., A.C., N.F. and J.C.C. designed the experiments; J.T.C., A.C., N.F., H.G.-R., R.T.B., A.T., C.M.S., S.D.G. and T.A.M. performed the experiments; J.T.C., A.C., N.F., S.D.G., T.A.M. and J.C.C. analyzed the data; J.T.C., A.C., N.F. and J.C.C. wrote the paper.

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Correspondence to James C Carrington.

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Cuperus, J., Carbonell, A., Fahlgren, N. et al. Unique functionality of 22-nt miRNAs in triggering RDR6-dependent siRNA biogenesis from target transcripts in Arabidopsis. Nat Struct Mol Biol 17, 997–1003 (2010). https://doi.org/10.1038/nsmb.1866

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