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| The EMBO Journal
(2000) 19, 997–1009, doi:10.1093/emboj/19.5.997 |
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| RNA recognition by a Staufen double-stranded RNA-binding domain |
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| Andres Ramos1, 2, 6, Stefan Grünert3, 4, 6, Jan Adams3, David R. Micklem3, Mark R. Proctor5, Stefan Freund5, Mark Bycroft5, Daniel St Johnston3 and Gabriele Varani1 |
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1 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK 2 Present address: Molecular Structure Division, NIMR, The Ridgeway, Mill Hill, London NW7 1AA, UK 3 Wellcome/CRC Institute and the Department of Genetics, University of Cambridge, Tennis Court Rd, Cambridge CB2 1QR, UK 4 Present address: IMP, Dr, Bohrgasse 7, 1030 Wien, Austria 5 Cambridge Centre for Protein Engineering, Hills Road, Cambridge CB2 2QH, UK 6 A.Ramos and S.Grünert contributed equally to this work To whom correspondence should be addressed Gabriele Varani, gv1@mrc-lmb.cam.ac.uk Received 25 November 1999; Revised 21 December 1999; Accepted 12 January 2000. |
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| Abstract |
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The double-stranded RNA-binding domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem–loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of bicoid and oskar mRNAs. Using high-resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem–loop. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix  1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix 1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins. |
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| Keywords: dsRBD, NMR, RNA localization, RNA–protein, Staufen |
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