Mechanism and Regulation of U1 snRNP Telescripting

项目摘要 这项提案的总体目标是了解一种新的主要基因- 表达过程，这是至关重要的全长转录的大多数蛋白质编码基因，并调节 人类和其他复杂生物体中的信使RNA（mRNA）亚型和mRNA长度。mrna被 从新生RNA聚合酶II（PolII）转录物加工，其通常包括去除内含子 （剪接）和转录终止3 '端切割和聚腺苷酸化（CPA）。拼接和CPA是 分别由剪接位点和CPA信号（PAS）指定。然而，许多PAS无法区分 从根本上说，基因末端的PAS分散在整个前体mRNA中，特别是在内含子和3'端， 非翻译区（3 'UTR），并且可以触发早熟CPA（PCPA）。PCPA被U1 snRNP抑制 (U1)，人类细胞中最丰富的非编码核RNA蛋白质小颗粒。为了简洁， 为了将其与U1在剪接中的作用区分开来，我们将U1对PCPA的抑制称为剪接（因为这是必要的）。 长距离转录）。与U1在剪接中的功能一样，剪接也依赖于U1 snRNA碱基配对 新生的转录物，可以废除与U1反义寡核苷酸（U1 AMO），导致PCPA。 最近的研究表明，即使U1和PAS之间的平衡发生微小变化， 基因表达，并能深刻地改变细胞产生的mRNA和蛋白质。这种变化发生在 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产生更短的mRNA亚型，需要响应 剧烈的环境变化。重要的是，U1 AMO重现了相同的mRNA亚型变化，U1过度表达。 表达可以阻止它们在受刺激的细胞中产生。U1 AMO也引起广泛的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转录在转录组调控中的首要作用影响转录、剪接、CPA， mRNA生命中的所有下游事件。它在生物学和医学上有许多潜在的应用。 要实现这一点，需要对U1抑制PAS的分子机制有详细的了解 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和切割及多聚腺苷酸化因子的转录组结合位置 （CPAF），并解释了它们与PCPA的位置。我们还抓获了U1和CPAF 复合物在细胞中，确定其组成和化学计量，并确定细胞如何产生 需要大量的U1来进行转录。这些进展为今后的研究奠定了基础。我 预计机制研究和新信息将确定潜在的干预点，包括 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.