The Fate of dsRNA in the Nucleus

描述（由申请人提供）：我们对细胞如何区分编码新蛋白质亚型的选择性编辑mrna和编码无功能突变蛋白质的dsrna诱导的混杂编辑rna感兴趣。我们已经发现了一种新的多蛋白复合物，它可以与含有肌苷的rna特异性地合作结合。该复合物包含肌苷特异性RNA结合蛋白p54nrb、剪接因子PSF和内核基质结构蛋白matrin 3。这项提议的总体目标是更多地了解这种复合物及其体内靶点。在第一个目标中，我们将表征I-RNA结合复合物。我们将确定p54nrb的哪些蛋白质结构域对各种生物功能至关重要，以及与RNA， DNA和其他蛋白质相互作用的重要序列。此外，我们将开展旨在表征p54nrb、PSF和matrin 3可能的体内功能的实验。

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.