Functional parameters of Dicer-independent microRNA biogenesis
- 1Department of Developmental Biology, Sloan-Kettering Institute, New York, New York 10065, USA
- 2Molecular Biology Program, Weill Graduate School of Medical Sciences, Cornell University, New York, New York 10065, USA
Abstract
Until recently, a Dicer-class RNase III enzyme was believed to be essential for microRNA (miRNA) biogenesis in all animals. The conserved vertebrate locus mir-451 defies this expectation and instead matures by direct cleavage of its pre-miRNA hairpin via the Slicer activity of Argonaute2 (Ago2). In this study, we used structure–function analysis to define the functional parameters of Ago2-mediated miRNA biogenesis. These include (1) the requirement for base-pairing at most, but not all, positions within the pre-mir-451 stem; (2) surprisingly little influence of the 5′-nucleotide on Ago sorting; (3) substantial influence of Ago protein stoichiometry on mir-451 maturation; (4) strong influence of G:C content in the distal stem on 3′ resection of cleaved mir-451 substrates; and (5) the influence of hairpin length on substrate utilization by Ago2 and Dicer. Unexpectedly, we find that certain hairpin lengths confer competence to mature via both Dicer-mediated and Ago2-mediated pathways, and we show, in fact, that a conventional shRNA can traverse the Dicer-independent pathway. Altogether, these data inform the design of effective Dicer-independent substrates for gene silencing and reveal novel aspects of substrate handling by Ago proteins.
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Footnotes
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↵3 Corresponding author.
E-mail laie{at}mskcc.org.
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.032938.112.
- Received February 16, 2012.
- Accepted February 29, 2012.
- Copyright © 2012 RNA Society
Freely available online through the RNA Open Access option.