The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1
- Biology Department and Huck Institutes of Life Sciences, Pennsylvania State University, University Park, PA 16802
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Contributed by Nina Fedoroff, May 1, 2008 (received for review March 30, 2008)
Abstract
The results of genetic studies in Arabidopsis indicate that three proteins, the RNase III DICER-Like1 (DCL1), the dsRNA-binding protein HYPONASTIC LEAVES1 (HYL1), and the C2H2 Zn-finger protein SERRATE (SE), are required for the accurate processing of microRNA (miRNA) precursors in the plant cell nucleus. To elucidate the biochemical mechanism of miRNA processing, we developed an in vitro miRNA processing assay using purified recombinant proteins. We find that DCL1 alone releases 21-nt short RNAs from dsRNA as well as synthetic miR167b precursor RNAs. However, correctly processed miRNAs constitute a minority of the cleavage products. We show that recombinant HYL1 and SE proteins accelerate the rate of DCL1-mediated cleavage of pre- and pri-miR167b substrates and promote accurate processing.
Footnotes
- ¶To whom correspondence should be addressed. E-mail: nvf1{at}psu.edu
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Author contributions: Z.D., M.-H.H., and N.F. designed research; Z.D. and M.-H.H. performed research; Z.D. and N.F. analyzed data; and Z.D. and N.F. wrote the paper.
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↵†Present address: Department of Molecular Biology, Cell Biology, and Biochemistry, Brown Medical School, Providence, RI 02912.
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↵‡Present address: Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208-3500.
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↵§Present address. Department of State, 2101 C Street NW, Washington, DC 20520.
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The authors declare no conflict of interest.
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See Commentary on page 9851.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0803356105/DCSupplemental.
- © 2008 by The National Academy of Sciences of the USA
