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

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。这笔赠款将详细介绍旨在了解这些过程的子集、前mRNA 3‘末端处理和siRNA生产的实验，以及它们通过一种重要的支架蛋白Symplekin在黑腹果蝇中的潜在联系。所有真核生物的前mRNAs都需要对其3‘端进行加工才能形成成熟的mRNAs。依赖于复制的组蛋白前mRNAs只在3‘端被切割，而所有其他真核生物的mRNAs都需要切割，然后再进行多聚腺苷酸化才能形成成熟的信息。有趣的是，尽管3‘端加工的这种变异导致了许多顺式和反式作用因子的不同，但最近在果蝇中的数据表明，所有真核细胞mRNAs的3’端加工是由相同的蛋白质复合体执行的。这个复合体是核心切割复合体，包含三种蛋白质：CPSF73、CPSF100和Symplekin。虽然我们了解了这种复合体的生物学作用，但它的组装和活性的分子要求还没有确定。最近，一类新的生物分子，小RNA，已经出现，对正常的细胞功能是不可或缺的。这些小干扰或微RNAs(分别是siRNAs或miRNAs)来自基因组和细胞外来源，并与较大的RNA分子相互作用，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，上一段描述的核心切割复合体的基本成分Symplekin与另一种蛋白质DICER-2相互作用，从较大的前体RNA中切割siRNA。虽然我之前与Symplekin的工作详细说明了它在前mRNA 3‘末端处理中的重要性，但它参与siRNA成熟途径是一个新的发现。具体地说，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mRNA 3‘末端处理和siRNA生物发生之间的潜在联系。我们假设Symplekin参与了siRNA的生物发生，并且果蝇的siRNA产生途径通过Symplekin与前mRNA 3‘末端处理密切相关。这项拨款中详述的实验将检验这一假设。

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.