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）：我们对细胞如何区分编码新蛋白质同工型的选择性编辑的mRNA和dsRNA诱导的，杂乱地编辑的RNA，该RNA编码非功能性突变蛋白。我们发现了一种新型的多蛋白质复合物，该复合物特异性地结合了含肌苷的RNA。该复合物包含肌苷特异性RNA结合蛋白p54NRB，剪接因子PSF和内部核基质基质结构蛋白质蛋白3。该建议的总体目标是更多地了解该复合物及其体内靶标。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的蛋白质结构域对于各种生物学功能至关重要，并且序列对于与RNA，DNA和其他蛋白质相互作用很重要。此外，我们将进行旨在表征P54NRB，PSF和Matrin 3的体内功能的实验。 最后，我们将融合P54NRB，其伴侣和突变体与MS2外套蛋白或噬菌体lambda N蛋白，生成结合除I-RNA以外的特定RNA序列的蛋白质。这些实验将提供一个系统，我们可以专门靶向要保留在核中的RNA。在第二个目标中，我们将确定P54NRB和复合物的体内靶标。许多方法将用于表征结合p54NRB或复合物的细胞RNA。这项工作还涉及使用小型双工RNA（siRNA）来抑制内源性P54NRB或ADAR活性。这些可能揭示了自然受反义RNA调节的新型细胞RNA。最后，将检查保留在原子核中的几个众所周知的聚腺苷酸化RNA，以结合p54NRB或较大复合物的能力。在最终目标中，我们将表征包括果蝇，chiromus和xenopus在内的其他生物中的I-RNA结合活性。这些研究可能会揭示这些蛋白质的重要​​和保守功能。对于每个生物体，我们将研究I-RNA结合蛋白及其分子伴侣。