The Fate of dsRNA in the Nucleus

描述(由申请人提供)：我们感兴趣的是细胞如何区分选择性编辑的mRNA和dsRNA诱导的、混杂编辑的RNA，前者编码新的蛋白质亚型，后者编码无功能的突变蛋白质。我们发现了一种新的多蛋白复合体，它能与含有肌苷的RNA特异性地和协同性地结合。该复合体包含肌苷特异性RNA结合蛋白p54nrb、剪接因子PSF和内核基质结构蛋白Matrin 3。本提案的总体目标是了解更多关于该复合体及其体内靶点的信息。在第一个目标中，我们将表征i-RNA结合复合体。我们将确定p54nrb的哪些蛋白质结构域对各种生物学功能至关重要，以及对其与RNA、DNA和其他蛋白质相互作用重要的序列。此外，我们还将进行实验，以确定p54nrb、PSF和Matrin 3在体内可能的功能。 最后，我们将p54nrb及其合作伙伴和突变体与MS2外壳蛋白或噬菌体lambda N蛋白融合，产生与I-RNA以外的特定RNA序列结合的蛋白质。这些实验将提供一个系统，在其中我们可以特定地靶向保留在细胞核中的RNA。在第二个目标中，我们将确定p54nrb及其复合体的体内靶点。将使用许多方法来表征结合p54nrb或复合体的细胞RNA。这项工作还将涉及使用小双链RNA(SiRNAs)来抑制内源性p54nrb或ADAR活性。这些可能揭示了天然受反义RNA调控的新型细胞RNA。最后，将检查几个保留在细胞核中的众所周知的聚腺苷酸化RNA与p54nrb或更大的复合体结合的能力。在最终目标中，我们将表征I-RNA在其他生物中的结合活性，包括果蝇、摇蚊和爪哇。这类研究可能有助于揭示这些蛋白质的重要和保守功能。对于每种生物，我们将研究i-RNA结合蛋白及其分子伴侣。

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.