The role of U1 snRNP proteins in snRNP biogenesis and gene expression regulation

项目总结 这项建议的长期目标是了解U1小核的调控机制 核糖核蛋白(U1)在核糖核蛋白生物发生中的作用及其在蛋白编码基因调控中的作用U1 SnRNP， 剪接体SnRNP中最丰富的RNP颗粒，在切除内含子(剪接)中起着关键作用。 以及防止提前终止新的成绩单(电传)。这两项活动都由 RNA：RNA碱基配对-U1 SnRNA和5‘剪接位点(5’ss)之间的配对。泛癌患者具有多发性 U1单链RNA的突变，包括5‘ss和U1单链RNP特异的蛋白结合序列，导致 通过新的5‘s识别实现癌基因和肿瘤抑制基因的选择性剪接。然而，我们的 初步数据显示，这些U1单链RNA突变不会形成稳定的Sm核心，而Sm核心是关键的中间体 由SMN(运动神经元存活)复合体组装，它决定了细胞的稳定性和丰度 U1SnRNP在细胞内表达。此外，我们发现了一种U1SnRNP特异的U1C蛋白，以前已知它是稳定的 5‘SS：U1 SNRNA碱基配对，在调节所有SnRNA的Sm核心组装中起着关键作用。目标是 这一建议的目的是研究新发现的U1C在SNRNP中作为守门人的作用机制 剪接体的生物发生及其对质量控制的潜在贡献和调控机制 拼接和远程脚本活动。该项目将同时解决U1C的两个功能，通过利用 两个已确定的成分剖析了U1C在SNRNP生物发生中的作用及其参与内含子的作用 多聚腺苷酸化和3‘非编码区长度的变化，癌基因在癌症中的分子特征。我们建议 追求三个具体目标：1)阐明带有SMN复合体的U1C在SnRNP中作为守门人的作用 生物发生学。2)研究泛癌患者中发现的U1SnRNA突变对Sm核心组装的影响。 3)研究远端转录中U1C与mRNA 3‘端终止机制的相互作用。使用生物化学 方法，我们将研究U1C-SMN复合体与U1C-U1单链RNA之间的分子联系。 通过检查这些联系，我们可以更好地了解U1C如何控制SnRNP曲目和 最终影响剪接体的功能。此外，我们还将描绘U1C与mRNA 3‘-的相互作用- 端面加工机械。这种相互作用是一个关键的开关，可以将U1 SnRNP从生产性剪接转化为 由于5‘s结合的丧失，导致mRNA过早终止或3’端非编码区缩短。为了阐明多个- U1C的刻面调控机制，我们将在体外采用Sm核心组装、RNA亲和纯化、 体外信使核糖核酸加工分析和细胞内甲醛交联与免疫沉淀联用 方法：研究方法。该项目的预期结果将确定U1C作为一种在SNRNP生物发生和mRNA中的作用 代谢，从而解释U1单链RNA突变在泛癌中的后果，并提供证据 通过失去端粒酶活性来进行基因调控。

PROJECT SUMMARY The long-term goal of this proposal is to understand the regulatory mechanisms of U1 small nuclear ribonucleoprotein (U1 snRNP) in snRNP biogenesis and its role in protein-coding gene regulation. U1 snRNP, the most abundant RNP particle among the spliceosomal snRNP, plays a key role in excising introns (splicing) and preventing premature termination of nascent transcripts (telescripting). Both activities are ensured by the RNA:RNA base-pairing between U1 snRNA and 5’ splice site (5’ss). Pan-cancer patients possess multiple mutations in the U1 snRNA, including 5’ss and U1 snRNP-specific protein binding sequences, which result in alternative splicing of oncogenes and tumor suppressors through novel 5’ss recognition. However, our preliminary data showed that those U1 snRNA mutations do not form a stable Sm core, a key intermediate assembled by the SMN (survival of motor neuron) complex, which determines the stability and abundance of the U1 snRNP in cells. Moreover, we identified that a U1 snRNP-specific U1C protein, previously known to stabilize 5’ss:U1 snRNA base-pairing, plays a critical role in regulating Sm core assembly of all snRNAs. The objective of this proposal is to investigate the mechanism of the newly discovered role of U1C as a gatekeeper in snRNP biogenesis, its potential contribution to the quality control of spliceosomes, and the regulatory mechanism in splicing and telescripting activity. This project will simultaneously address both functions of U1C by harnessing two established components to dissect the role of U1C in snRNP biogenesis as well as its involvement in intronic polyadenylation and 3’UTR length changes, molecular characteristics of the oncogenes in cancer. We propose to pursue three specific aims: 1) Elucidate the role of U1C with SMN complex as a gatekeeper in snRNP biogenesis. 2) Investigate the effects of U1 snRNA mutations found in pan-cancer patients on Sm core assembly. 3) Examine the U1C interaction with mRNA 3’-end termination machinery in telescripting. Using biochemical approaches, we will investigate the molecular connections between U1C-SMN complex and U1C-U1 snRNA. By examining these connections, we can better understand how U1C controls the snRNP repertoire and ultimately impact the function of spliceosome. Moreover, we will delineate the U1C’s interaction with mRNA 3’- end processing machinery. This interaction is a key switch that can convert U1 snRNP from productive splicing to premature mRNA termination or 3’UTR shortening through the loss of 5’ss binding. To elucidate the multi- faceted regulation mechanism of U1C, we will employ in vitro Sm core assembly, RNA-affinity purification, in vitro mRNA processing assays, and in-cell formaldehyde-crosslinking coupled with immunoprecipitation methods. The expected outcome of this project will identify the role of U1C as a in snRNP biogenesis and mRNA metabolism, thus explaining the consequence of the U1 snRNA mutations in pan-cancer and providing evidence of gene regulation by the loss of telescripting activity.