The Fate of dsRNA in the Nucleus

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DESCRIPTION (provided by applicant): We are interested in how cells discriminate between selectively edited mRNAs that encode new protein isoforms, and dsRNA-induced, promiscuously-edited RNAs that encode nonfunctional, mutant proteins. We have discovered a novel multiprotein complex that binds specifically and cooperatively to inosine-containing RNAs. This complex contains the inosine-specific RNA binding protein p54nrb, the splicing factor PSF and the inner nuclear matrix structural protein matrin 3. This overall goal of this proposal is to learn more about this complex and its in vivo targets. In the first aim we will characterize the I-RNA binding complex. We will determine which protein domains of p54nrb are critical for various biological functions, and sequences important for its interaction with RNA, DNA and other proteins. In addition, we will carry out experiments designed to characterize possible in vivo functions of p54nrb, PSF and matrin 3. Finally, we will fuse p54nrb, its partners and mutants to the MS2 coat protein or phage lambda N protein, generating proteins that bind specific RNA sequences other than I-RNA. These experiments will provide a system where we can specifically target RNAs to be retained in the nucleus. In the second aim we will identify in vivo targets for p54nrb and the complex. A number of approaches will be used to characterize cellular RNAs that bind p54nrb or the complex. This work wil also involve the use of small duplex RNAs (siRNAs) to inhibit endogenous p54nrb or ADAR activity. These might reveal novel cellular RNAs that are naturally regulated by antisense RNA. Finally, several well-known polyadenylated RNAs that are retained in the nucleus will be examined for their ability to bind p54nrb or the larger complex. In the final aim we will characterize the I-RNA binding activities in other organisms, including Drosophila, Chironomus and Xenopus. Such studies might shed light on vital and conserved functions for these proteins. For each organism, we will study the I-RNA binding protein and its molecular partners.