Genetics and Genomics of Alternative Polyadenylation and miRNA Regulation in C. e - Renewal - 1

摘要 3'非翻译区（3' UTR）是位于终止密码子和polyA尾之间的mRNA末端。他们 包含许多缺乏特征的调控元件，使其成为后- miRNA和RNA结合蛋白对基因表达的转录调控。miRNAs很小 结合3 'UTR并抑制mRNA翻译的调节RNA。我们的团队和其他人已经证明， 选择性多聚腺苷酸化（阿帕），在后生动物中观察到的一个难以捉摸的过程， 各种3 'UTR同种型广泛存在。虽然阿帕经常出现在正常状态和几乎所有 疾病，它在细胞和组织中的生物学作用是一个谜。具有较长3 'UTR的基因具有更大的潜力 调控位点，因此阿帕可能允许基因改变其3 'UTR长度并逃避miRNAs的抑制 和RNA结合蛋白。由于阿帕是丰富的，这可以解释为什么没有直接的相关性 细胞转录组和蛋白质组之间的联系。了解3 '端形成的整体机制，阿帕 功能，和miRNA靶向将推进转录后修饰的研究。后生动物 拥有数百种miRNAs和数千种3 'UTR亚型，其中大多数在细胞中表达和发挥作用。 不同的组织和发育环境。我们对这些网络的运作方式知之甚少， 尽管它们在控制基因表达方面起着关键作用，但它们彼此之间却相互交流。我们需要获得新的 数据和见解，并通过获取和融合组织- 特定的miRNA定位数据、miRNA靶数据和3 'UTR亚型数据。我们的Interactome将提供 在体内转录后基因调控功能的全球基础，阐明其在正常和 多细胞动物的疾病状态。在我们的第一个R 01基金中，我们研究了组织特异性阿帕事件和miRNA网络 线虫C. elegans，以定义和探测蠕虫miRNAs/3 'UTR相互作用组。我们鉴定了3 'UTR 8种C. elegans体细胞组织，获得了三个最大的和 疾病相关C. elegans蠕虫组织，开发并实施了无偏的方法来绘制miRNA 高通量的目标，并在我们的研究中确定的基因中启动阿帕选择的机制研究。 对于这个新的提交，我们将：（1）通过包括新的组织特异性3 'UTR来完成蠕虫3' UTR组 同种型，（2）鉴定八种蠕虫体细胞组织中的组织特异性miRNA群体，以及（3） 机制研究，以确定组织特异性PAS的选择，在阿帕的背景下。我们精心策划的3 'UTRome将 是第一个提供完整的后生动物3 'UTRome的数据库，以单一的基本分辨率绘制。 研究PAS选择的三个全局假设可以揭示全局转录后基因调控 组织发育和疾病中涉及的机制，因此具有高影响。我们认为 这些新研究的影响很大，因为它们将为全球理解提供重要结果， 后生动物转录后基因调控生物学。 1

Abstract 3’ Untranslated Regions (3’UTR) are mRNA ends located between the STOP codon and the polyA tail. They contain numerous regulatory elements that are poorly characterized, making them a core target for post- transcriptional regulation of gene expression by miRNAs and RNA binding proteins. MiRNAs are small regulatory RNAs that bind 3’UTRs and repress mRNA translation. Our group and others have shown that alternative polyadenylation (APA), an elusive process observed in metazoans that gives rise to genes with various 3’UTR isoforms, is widespread. While APA is frequently present in normal states and in virtually all diseases, its biological role in cells and tissues is a mystery. Genes with longer 3’UTRs have more potential regulatory sites, thus APA may allow genes to alter their 3’UTR length and escape the repression by miRNAs and RNA binding proteins. Since APA is abundant this could explain why there is not a direct correlation between cellular transcriptomes and proteomes. Understanding the global mechanics of 3’end formation, APA function, and miRNA targeting would advance research on post-transcriptional modifications. Metazoans possess hundreds of miRNAs and thousands of 3’UTR isoforms, most of which are expressed and function in different tissues and developmental contexts. We know little about how these networks operate and communicate with each other, despite their critical role in controlling gene expression. We need to gain new data and insights, and develop a high-resolution miRNAs/3’UTRs Interactome by acquiring and fusing tissue- specific miRNA localization data, miRNA target data, and 3’UTRs isoform data. Our Interactome will provide the global basis of post-transcriptional gene regulation function in vivo, elucidating its role in both normal and disease states in metazoans. In our first R01 grant we studied tissue-specific APA events and miRNA networks in the nematode C. elegans, to define and probe the worm miRNAs/3’UTRs Interactome. We identified 3’UTR isoforms in eight C. elegans somatic tissues, acquired miRNA expression data for the three largest and disease-relevant C. elegans worm tissues, developed and implemented unbiased methods to map miRNA targets in high throughput, and initiated mechanistic studies of APA selection in genes identified in our studies. For this new submission we will, (1) complete the worm 3’UTRome by including new tissue-specific 3’UTR isoforms, (2) identify tissue-specific miRNA populations in eight worm somatic tissues and, (3) perform mechanistic studies to determine tissue-specific PAS choice in the context of APA. Our curated 3’UTRome will be the first database to provide a complete metazoan 3’UTRome mapped at a single base resolution. Investigating three global hypotheses of PAS choice can uncover global post-transcriptional gene regulatory mechanisms implicated in tissue development and disease, and therefore of high impact. We believe that the impact of these new studies is high since will provide important results toward a global understanding the biology of post-transcriptional gene regulation in metazoans. 1