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The link between mRNA 3' end processing and siRNA biogenesis in Drosophila

果蝇中 mRNA 3 末端加工与 siRNA 生物合成之间的联系

基本信息

批准号：
8574991
负责人：
Mindy Steiniger
金额：
$ 32.96万
依托单位：
UNIVERSITY OF MISSOURI-ST. LOUIS
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-16 至 2017-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8574991
关键词：
Affect Binding Biogenesis Biological Cell physiology Cells Cleaved cell Co-Immunoprecipitations Complex Confocal Microscopy Data Disease Double-Stranded RNA Drosophila genus Drosophila melanogaster Event Fractionation Gene Expression Regulation Generations Genetic Transcription Genomics Goals Grant Heterochromatin Histones Knowledge Link Location Malignant Neoplasms Messenger RNA Metabolic Pathway Metabolism MicroRNAs Modification Molecular Nuclear Pathway interactions Poly A Polyadenylation Precursor RNA Process Production Proteins RNA RNA Interference RNA Processing Relative (related person)Research Ribosomal RNA Role Scaffolding Protein Small Interfering RNA Small RNA Source Testing Trans-Activators Transfer RNA Translations Two-Hybrid System Techniques Variant Work Yeasts abstracting chromatin immunoprecipitation deep sequencing design extracellular human DICER1 protein in vitro Assay in vivo mRNA Precursor mRNA capping mutant novel protein complex protein protein interaction protein transport public health relevance research study tissue culture trafficking

项目摘要

DESCRIPTION (provided by applicant): Abstract RNA processing has emerged as an important step in post-transcriptional gene regulation. This grant will detail experiments designed to understand a subset of these processes, pre-mRNA 3' end processing and siRNA production, and their potential link via an important scaffolding protein, Symplekin, in Drosophila melanogaster. All eukaryotic pre-mRNAs require processing of their 3' ends to form mature mRNAs. Replication-dependant histone pre-mRNAs are only cleaved at the 3' end, while all other eukaryotic mRNAs require cleavage followed by polyadenylation to form a mature message. Interestingly, even though this variation in 3' end processing necessitates differences in many cis and trans acting factors, recent data in Drosophila indicate that 3' end processing of all eukaryotic mRNAs is performed by the same protein complex. This complex, the core cleavage complex, contains three proteins: CPSF73, CPSF100 and Symplekin. While we understand the biological role of this complex, the molecular requirements for its assembly and activity have not been determined. Recently, a new class of biomolecules, small RNAs, has emerged as being integral for proper cellular function. These small interfering or micro RNAs (siRNAs or miRNAs, respectively) are derived from both genomic and extracellular sources and interact with larger RNA molecules to affect downstream cellular processes such as transcription, translation and heterochromatin formation. Our preliminary experiments suggest that Symplekin, the essential component of the core cleavage complex described in the previous paragraph, interacts with another protein, Dicer-2, to cut siRNAs from larger precursor RNAs. While my previous work with Symplekin detailed its importance in pre- mRNA 3' end processing, its involvement in the siRNA maturation pathway is a novel discovery. Specifically, we aim to characterize molecular interactions in the core cleavage factor, determine the role of Symplekin in Drosophila siRNA metabolism and investigate the potential link between pre-mRNA 3' end processing and siRNA biogenesis. We hypothesize that Symplekin participates in siRNA biogenesis and that the siRNA production pathway in Drosophila is intimately connected to pre-mRNA 3' end processing via Symplekin. Experiments detailed in this grant will test this hypothesis.

描述（由申请人提供）：摘要RNA加工已成为转录后基因调控的重要步骤。该资助将详细介绍旨在了解这些过程的一个子集、前mRNA 3'末端加工和siRNA产生的实验，以及它们通过果蝇中重要的支架蛋白Symplekin的潜在联系。所有真核前体mRNA都需要加工其3'末端以形成成熟mRNA。复制依赖性组蛋白前体mRNA仅在3'末端被切割，而所有其他真核mRNA需要切割，然后进行聚腺苷酸化以形成成熟的信使。有趣的是，尽管3'端加工的这种变化需要许多顺式和反式作用因子的差异，但果蝇中最近的数据表明，所有真核生物mRNA的3'端加工都是由相同的蛋白质复合物进行的。这个复合物，核心切割复合物，包含三种蛋白质：CPSF 73，CPSF 100和Symplekin。虽然我们了解这种复合物的生物学作用，但其组装和活性的分子要求尚未确定。最近，一类新的生物分子，小RNA，已经成为适当的细胞功能的组成部分。这些小干扰或微RNA（分别为siRNA或miRNA）来源于基因组和细胞外来源，并与较大的RNA分子相互作用以影响下游细胞过程，如转录、翻译和异染色质形成。我们的初步实验表明，Symplekin是前一段中描述的核心切割复合物的重要组成部分，它与另一种蛋白Dicer-2相互作用，从较大的前体RNA中切割siRNA。虽然我以前对Symplekin的研究详细描述了它在前mRNA 3'末端加工中的重要性，但它参与siRNA成熟途径是一个新的发现。具体而言，我们的目标是表征核心切割因子中的分子相互作用，确定Symplekin在果蝇siRNA代谢中的作用，并研究前体mRNA 3'末端加工和siRNA生物合成之间的潜在联系。我们假设Symplekin参与siRNA的生物合成，并且果蝇中的siRNA产生途径与通过Symplekin的前mRNA 3'末端加工密切相关。本资助中详细描述的实验将验证这一假设。