Sex-lethal Interactions with Protein and RNA
ROLES OF GLYCINE-RICH AND RNA BINDING DOMAINS*
- From the Molecular Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-1340
Abstract
Sex-lethal (Sxl) is an RNA-binding protein, containing two conserved RNA binding domains (RBDs) and a glycine-rich region, which functions as a regulator of alternative splicing inDrosophila sex determination. Previous work demonstrated that Sxl monomers interact cooperatively upon binding to target RNAs and that the cooperativity depends on the glycine-rich N terminus. Here we use band shift experiments to show that RNA binding patterns are altered when Sxl is combined with other proteins having similar glycine-rich domains, including mammalian heterogeneous nuclear (hn) RNP L and Drosophila Hrb87F (an hnRNP A/B homolog). Direct involvement of the Sxl glycine-rich region in protein interactions was verified by Far-Western analysis. Two interaction domains, the Sxl N terminus and the Sxl first RNA binding domain, were suggested by the yeast two-hybrid assay. In a systematic examination of the RNA binding properties of Sxl domains, it was found that the Sxl termini as well as the RBDs influence RNA binding specificity. Finally, selection of the Sxl optimal binding site (SELEX) confirms the importance of U-runs in the Sxl binding site and suggests a second type of non-U-run target that may be associated with RNA secondary structure.
Footnotes
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↵* This work was supported by a grant from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Present address: Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093-0651.
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↵§ To whom correspondence should be addressed. Tel.: 213-740-5197; Fax: 213-740-8631; E-mail: LBELL{at}mizar.usc.edu.
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↵1 The abbreviations used are: RBD, RNA binding domain; Sxl, sex-lethal; RNP, ribonucleoprotein; PCR, polymerase chain reaction; aa, amino acids; bp, base pairs; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; hn, hetorogeneous nuclear; sn, small nuclear.
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- Received December 3, 1996.
- Revision received June 25, 1997.
