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A micrococcal nuclease homologue in RNAi effector complexes

Abstract

RNA interference (RNAi) regulates gene expression by the cleavage of messenger RNA, by mRNA degradation and by preventing protein synthesis. These effects are mediated by a ribonucleoprotein complex known as RISC (RNA-induced silencing complex)1. We have previously identified four Drosophila components (short interfering RNAs1, Argonaute 2 (ref. 2), VIG and FXR3) of a RISC enzyme that degrades specific mRNAs in response to a double-stranded-RNA trigger. Here we show that Tudor-SN (tudor staphylococcal nuclease)—a protein containing five staphylococcal/micrococcal nuclease domains and a tudor domain—is a component of the RISC enzyme in Caenorhabditis elegans, Drosophila and mammals. Although Tudor-SN contains non-canonical active-site sequences, we show that purified Tudor-SN exhibits nuclease activity similar to that of other staphylococcal nucleases. Notably, both purified Tudor-SN and RISC are inhibited by a specific competitive inhibitor of micrococcal nuclease. Tudor-SN is the first RISC subunit to be identified that contains a recognizable nuclease domain, and could therefore contribute to the RNA degradation observed in RNAi.

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Figure 1: Identification and confirmation of Tudor-SN as a component of RISC complexes.
Figure 2: Immunoprecipitations from multiple organisms confirm association between Tudor-SN and components of RISC.
Figure 3: Tudor-SN has nuclease activity.
Figure 4: Staining of animals carrying a transgene expressing LacZ in the seam cells, under the translational control of let-7.

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Acknowledgements

We thank A. Mildvan, M. Tijsterman and T. Sijen for discussions. We thank T. Keenan for an anti-p100 antibody, T. Hobman for GERP (EIF2C2/hAgo2) antibody, H. Siomi for a FXR monoclonal antibody, and F. Slack for the lacZlin-41 reporter. A.A.C. is a George A. and Marjorie H. Anderson Fellow of the Watson School of Biological Sciences, and a Howard Hughes Medical Institute Predoctoral Fellow. A.M.D. is a David Koch Fellow of the Watson School of Biological Sciences. J.M.S. is supported by a postdoctoral fellowship from the US Army Prostate Cancer Research programme. G.J.H. is a Rita Allen Foundation Scholar and is supported by an Innovator Award from the US Army Breast Cancer Research programme. This work was also supported by a grant from the National Institutes of Health (G.J.H.) and by a VENI fellowship from the Netherlands Organization for Scientific Research (RFK).

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Correspondence to Gregory J. Hannon or Ronald H. A. Plasterk.

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Caudy, A., Ketting, R., Hammond, S. et al. A micrococcal nuclease homologue in RNAi effector complexes. Nature 425, 411–414 (2003). https://doi.org/10.1038/nature01956

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