Regulation of mRNA processing: Mechanisms and consequences

 描述(申请人提供)：在真核细胞中合成信使核糖核酸是一个高度复杂的过程，除了转录本身，还包括信使核糖核酸前体的剪接和3‘端形成。多年来，我的实验室一直在研究这些过程的机制和调控，还展示了它们如何与其他细胞事件(如DNA损伤)相结合，以及它们在分化和疾病中的作用。这个建议结合了我们在这些领域的研究，可以分为以下三个大的领域：1)mRNA的加工与疾病。将继续研究与RNA代谢有关的几种蛋白质在癌症和神经退行性疾病中的作用。RNA加工因子在癌症中的作用反映了两种不同的机制，即剪接调节蛋白的过度表达和编码各种剪接因子的基因突变。对于前者，正在进行的实验源于阐明癌症中PKM选择性剪接(AS)放松调控的潜在机制的研究。重要的问题包括机制的普遍性，剪接因子过度表达对肿瘤发展的重要性，以及关键调控剪接事件的识别。剪接因子突变可导致MDS和某些白血病，正在进行的研究旨在阐明突变影响几个基因功能的机制，例如SRSF2和SF3B1，以及这些缺陷是如何导致疾病的。在神经退行性疾病方面，工作重点是RNA/DNA结合蛋白TLS/FUS中导致ALS的突变如何扰乱蛋白质功能；C9ORF72中的六核苷酸重复扩增如何形成G-四链结构，从而隔离hnRNP H，破坏AS，并促进ALS；以及RNA/DNA解旋酶Senataxin的致病突变如何影响其在防止转录诱导的DNA损伤中的作用。2)基因的加工和分化。将继续研究AS和替代多聚腺苷酸化(APA)的变化如何有助于人类胚胎干细胞的多能性和分化。AS如何影响转录调控因子TCF3的功能，以及这如何促进分化将被确定。AS事件的机制，包括剪接调节器本身是如何受到监管的，将被调查。我们意想不到的发现是，编码两个PA因子的转录产物在分化过程中发生了变化，这两个转录因子都是CPSF的亚基，并与AAUAAA识别有关。不同的异构体如何影响APA和分化，以及它们影响PA位点选择的机制将被确定。3)RNA加工和转录。检测RNA polII CTD功能的实验将继续进行，这是一个由26-52个七肽重复组成的独特结构域(共识YSPTSPS)。例如，最近的实验确定了P-Tyr1残基在促进某些lncRNA周转方面的作用，并表明 这涉及到与RNA解旋酶MTR4和PA机制的相互作用，将继续进行。这些实验和其他实验将促进我们对连接转录和RNA加工的“CTD代码”的理解。

 DESCRIPTION (provided by applicant): Synthesis of mRNAs in eukaryotic cells is a highly complex process, including, in addition to transcription itself, splicing and 3' end formation of mRNA precursors. My laboratory has studied the mechanisms and regulation of these processes for many years, and also shown how they are integrated with other cellular events such as DNA damage, and also how they function in differentiation and disease. This proposal combines our studies in these areas, and can be divided into the following three broad areas: 1) mRNA processing and disease. Studies examining the roles of several proteins implicated in RNA metabolism in cancer and neurodegenerative disease will be pursued. The role of RNA processing factors in cancer reflects two distinct mechanisms, overexpression of splicing regulatory proteins and mutation of genes encoding various splicing factors. With respect to the former, ongoing experiments stemming from studies elucidating the mechanism underlying deregulation of PKM alternative splicing (AS) in cancer will be pursued. Important questions include the generality of the mechanism, the importance of splicing factor overexpression to tumor development, and the identity of the critical regulated splicing events. Splicing factor mutations can cause MDS and certain leukemias, and ongoing studies are aimed at elucidating mechanisms by which mutations affect the function of several, e.g., SRSF2 and SF3B1, and how such defects lead to disease. With respect to neurodegenerative disease, work centers on how ALS-causing mutations in the RNA/DNA-binding protein TLS/FUS disrupt protein function; how the hexanucleotide repeat expansions in C9ORF72 form G-quadruplex structures that sequester hnRNP H, disrupt AS, and contribute to ALS; and how disease-causing mutations in the RNA/DNA helicase Senataxin affect its role in prevention of transcription-induced DNA damage. 2) mRNA processing and differentiation. Studies examining how changes in AS and alternative polyadenylation (APA) contribute to human embryonic stem cell pluripotency and differentiation will be pursued. How AS affects the function of the transcriptional regulator TCF3 and how this contributes to differentiation will be determined. The mechanism of the AS event, including how the splicing regulators are themselves regulated, will be investigated. Our unexpected finding that AS of transcripts encoding two PA factors, both subunits of CPSF and implicated in AAUAAA recognition, is altered during differentiation will be pursued. How the distinct isoforms affect APA, and differentiation, will be determined, as will the mechanism by which they influence PA site choice. 3) RNA processing and transcription. Experiments examining the function of the RNA pol II CTD, a unique domain consisting of 26-52 heptad repeats (consensus YSPTSPS), will be continued. For example, recent experiments establishing a role for P-Tyr1 residues in facilitating turnover of certain lncRNAs, and suggesting that this involves interaction with the RNA helicase Mtr4 and the PA machinery, will be pursued. These and other experiments will advance our understanding of the "CTD code" that links transcription and RNA processing.