The Fate of dsRNA in the Nucleus

dsRNA 在细胞核中的命运

• 批准号: 6557232
• 负责人: Gordon G Carmichael
• 金额: $ 25.38万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6557232
• 关键词: Chironomidae; Drosophilidae; RNA binding protein; SDS polyacrylamide gel electrophoresis; Xenopus; cell nucleus; double stranded RNA; gene expression; polymerase chain reaction; protein binding; protein protein interaction; protein purification; protein structure function; serial analysis of gene expression; thin layer chromatography; tissue /cell culture; western blottings

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.

描述（由申请人提供）：我们感兴趣的是细胞如何区分编码新蛋白质同种型的选择性编辑的mRNA和编码非功能性突变蛋白质的dsRNA诱导的混杂编辑的RNA。我们已经发现了一种新的多蛋白复合物，它特异性地与含肌苷的RNA协同结合。该复合物包含肌苷特异性RNA结合蛋白p54 nrb、剪接因子PSF和内核基质结构蛋白matrin 3。该提案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p54 nrb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，我们将进行旨在表征p54 nrb，PSF和matrin 3可能的体内功能的实验。 最后，我们将融合p54 nrb，它的合作伙伴和突变体的MS 2外壳蛋白或噬菌体λ N蛋白，产生的蛋白质，结合特定的RNA序列以外的I-RNA。这些实验将提供一个系统，我们可以特异性地靶向RNA保留在细胞核中。在第二个目标中，我们将确定p54 nrb和复合物的体内靶点。许多方法将用于表征结合p54 nrb或复合物的细胞RNA。这项工作还将涉及使用小双链RNA（siRNA）来抑制内源性p54 nrb或阿达尔活性。这些可能揭示了新的细胞RNA，自然地由反义RNA调节。最后，将检查保留在细胞核中的几种众所周知的多聚腺苷酸化RNA结合p54 nrb或更大复合物的能力。在最后的目标，我们将在其他生物，包括果蝇，摇蚊和爪蟾的I-RNA结合活动的特点。这些研究可能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。