Mechanism and Regulation of U1 snRNP Telescripting

项目总结 这项提议的总体目标是了解端粒转录的分子机制，这是一种新的和主要的基因 对大多数蛋白质编码基因的全长转录至关重要的表达过程，并调节 信使核糖核酸(MRNAs)在人类和其他复杂生物中的异构体和mRNA长度。MRNA是 从新生RNA聚合酶II(PolII)转录本加工而来，通常包括去除内含子 (剪接)和转录终止3‘端裂解和多聚腺苷化(CPA)。拼接和CPA是 分别由剪接点和CPA信号(通过)指定。然而，许多传球难以区分 从最终来说，基因末端的传递散布在前mRNAs中，特别是在内含子和3‘端。 非翻译区(3‘UTRs)，并可引发早产CPA(PCPA)。PCPA被U1 snRNP抑制 (U1)，人类细胞最丰富的非编码核小RNA-蛋白质颗粒。为简洁起见，并 区别于U1的S在剪接中的作用，我们称之为U1抑制PCPA，Telescriting(因为它是 远距离转录)。与U1在剪接中的功能一样，远程转录也依赖于U1的SnRNA碱基配对 可以被U1反义寡核苷酸(U1 AMO)废除，引起PCPA。 最近的研究表明，U1和PASS之间的平衡即使有微小的变化，也会对 基因表达，并可以深刻改变细胞产生的mRNAs和蛋白质。这样的变化就会发生 自然地，例如，在细胞刺激期间快速转录上调，并产生短暂的U1缺陷 相对于转录输出，导致PCPA产生需要反应的较短的mRNA异构体 剧烈的环境变化。重要的是，U1 AMO重现了相同的mRNA异构体移位，而U1过度- 表达可以阻止它们在刺激细胞中的产生。U1 AMO还引起广泛的3‘UTR缩短， 它发生在细胞增殖和癌症中，并有助于细胞增殖和癌症。 U1端粒转录在转录组调控中的主导作用影响转录、剪接、CPA，从而 MRNAs生命中的所有下游事件。它在生物学和医学方面有许多潜在的应用。 要实现它们，需要详细了解U1抑制PASS的分子机制 以及调节它的因素，这些因素仍然未知。我们在这方面取得了重大进展， 包括绘制U1转录组结合位置以及切割和多聚腺苷化因子的图谱 (CPAFs)，并解释了它们与PCPA地点的关系。我们还捕获了U1和CPAFs 细胞中的络合物，确定它们的组成和化学计量，并确定细胞如何产生 远程抄写需要大量的U1。这些进展为未来的研究奠定了基础。我 预计机制研究和新信息将确定潜在的干预点，包括 可药物靶点，并将推进利用它们进行新疗法的前景。

PROJECT SUMMARY This proposal’s overall goal is to understand the molecular mechanism of telescripting, a new and major gene expression process that is crucial for full-length transcription of most protein-coding genes and regulates messenger RNAs (mRNAs) isoforms and mRNA length in humans and other complex organisms. mRNAs are processed from nascent RNA polymerase II (PolII) transcripts, which generally includes the removal of introns (splicing) and transcription-terminating 3’-end cleavage and polyadenylation (CPA). Splicing and CPA are specified by splice sites and CPA signals (PASs), respectively. However, numerous PASs indistinguishable from the ultimate, gene ends’ PASs are scattered throughout pre-mRNAs, especially in introns and 3’ untranslated regions (3’UTRs), and can trigger premature CPA (PCPA). PCPA is suppressed by U1 snRNP (U1), human cells’ most abundant small non-coding nuclear RNA-protein particle. For brevity, and to distinguish it from U1’s role in splicing, we call U1 suppression of PCPA, telescripting (as it is necessary for long-distance transcription). Like U1 function in splicing, telescripting also depends on U1 snRNA base-pairing to nascent transcripts, which can be abrogated with U1 antisense oligonucleotides (U1 AMO), causing PCPA. Recent studies revealed that even slight changes in the balance between U1 and PASs has great impact on gene expression and can profoundly alter the mRNAs and proteins cells produce. Such changes occur naturally, for example rapid transcription up-regulation during cell stimulation, and create transient U1 deficit relative to transcription output, causing PCPA that produces shorter mRNA isoforms needed to respond to acute environmental changes. Importantly, U1 AMO recapitulates the same mRNA isoform shifts and U1 over- expression can prevent their production in stimulated cells. U1 AMO also elicits widespread 3'UTR shortening, which occurs in and contributes to cell proliferation and cancer. U1 telescripting’s overarching role in transcriptome regulation impacts transcription, splicing, CPA and thereby all downstream events in the life of mRNAs. It has numerous potential applications in biology and medicine. Realizing them requires detailed understanding of the molecular mechanism by which U1 suppresses PASs and the factors that regulate it, which remain unknown. We have made significant progress towards that, including mapping the transcriptome binding locations of U1 and cleavage and polyadenylation factors (CPAFs), and interpreted them in relation to PCPA locations. We have also captured U1 and CPAFs complexes in cells, determined their compositions and stoichiometries, and determined how cells produce the great U1 abundance required for telescripting. These advances lay the foundation for future studies. I anticipate the mechanistic studies and new information will identify potential points of intervention, including druggable targets, and will advance the prospects of harnessing them for novel therapies.