In silico Exploration of Alternative Polyadenylation Using Next-Generation Sequen

描述(由申请人提供)：本项目的总体目标是促进我们对真核生物中的替代聚腺苷酸化(APA)的理解，包括它发生的程度和它被调控的机制。真核基因的成熟需要几个转录后过程，其中之一就是3‘端的形成。这一过程包括Pre-mRNA的切割和多聚化，在Pre-mRNA的新切割的3‘端增加了一个聚(A)尾。它是由Pre-mRNA上的Poly(A)信号基序引导的。多聚腺苷酸化已被证明影响mRNA的稳定性、可译性和从细胞核到细胞质的转运。对于复杂的问题，许多真核基因都有不止一个Poly(A)位点。交替多聚腺苷化(APA)是一种利用这些不同的聚(A)位点来产生同一基因不同3‘端的mRNA转录本的现象。越来越多的证据表明，APA是调节基因表达的关键因素，影响编码蛋白的mRNA水平和/或功能。它还影响含有潜在顺式调控基序的3‘-UTR的性质和长度，这些基序对于mRNA的稳定性和翻译抑制是重要的。APA似乎同时受到发育和环境因素的调节，它通常以组织和/或疾病特有的方式发生。多聚(A)信号或多聚腺苷化蛋白因子的突变会导致包括癌症在内的严重疾病。然而，关于APA的潜在机制仍有许多重要的未解之谜。例如，尚不清楚发育和环境线索是如何被传导到APA过程的，也不清楚需要什么特殊的聚(A)信号(即特殊的RNA结构)来指导APA。下一代测序(NGS)使转录组测序发生了革命性的变化，使我们能够实现比桑格测序更高的序列深度和覆盖率。由此产生的大量NGS数据集使我们有可能将创新的新方法应用到我们的聚腺苷酸化生物信息学分析中，这将促进我们对APA及其调控机制的理解。具体地说，Poly(A)位点将根据它们的位置(例如，内含子与外显子内)、类型(例如，反义与正义)和生物学后果(例如，非正义介导的衰退、蛋白质产物截断和微小RNA结合位点的修饰)进行更准确的注释和分类。我们将研究APA模式和相关的顺式调控基序如何在不同物种、基因类型、组织、发育阶段和环境胁迫中发生变化。我们将通过湿法实验室方案来验证我们的预测结果。最后，我们将通过一个经过丰富分析和可视化的在线数据库和Web服务，将我们的数据、协议和软件发布给研究界。完成我们的模式生物这个项目的目标，将为更全面地了解人类和其他真核生物的这些复杂的调控机制铺平道路。 与公共健康相关：当真核基因表达时，它们的mRNAs必须在成熟过程中进行处理，其中一个步骤是聚腺苷酸化：连接一个聚(A)尾巴来标记和保护mRNA的末端。然而，位于不同多聚(A)位点的选择性多聚腺苷酸化(APA)会导致该mRNA的信息丢失，并与癌症和其他疾病有关。使用下一代测序数据，我们将采用创新的方法来准确地注释PolyA(A)位点，检测PolyA信号，研究APA及其调控机制，并为生物医学/生物研究团体提供丰富的信息数据库，以促进我们对APA在人类疾病中作用的理解。

DESCRIPTION (provided by applicant): The overall goal of this project is to further our understanding of alternative polyadenylation (APA) in eukaryotes, including the extent to which it occurs and the mechanisms by which it is regulated. Maturation of eukaryotic mRNA requires the actions of several post-transcriptional processes, one of which is 3'-end formation. This process consists of pre-mRNA cleavage and polydenylation, where a poly(A) tail is added to the newly cleaved 3'-end of pre-mRNA. It is guided by poly(A) signal motifs on the pre-mRNA. Polyadenylation has been shown to influence mRNA stability, translatability and transportability from the nucleus to cytoplasm. To complicated matters, many eukaryotic genes have more than one poly(A) site. Alternative polyadenylation (APA) is a phenomenon in which these different poly(A) sites are utilized to generate mRNA transcripts with different 3'-ends of the same gene. Increasing evidence suggests that APA is a key contributor in regulating gene expression, affecting mRNA levels and/or functions of coded proteins. It also affects the nature and length of the 3'-UTR harboring potential cis-regulatory motifs that are important for mRNA stability and translation suppression. APA appears to be regulated by both developmental and environmental cues, and it often occurs in a tissue- and/or disease-specific manner. Mutations of poly(A) signals or polyadenylation protein factors cause severe diseases including cancers. However, there are many important unanswered questions about underlying mechanisms of APA. For example, it is unclear how developmental and environmental cues are transduced to the APA process, or what special poly(A) signals (i.e., special RNA structures) are needed to guide APA. Next-Generation sequencing (NGS) has revolutionized transcriptome sequencing, allowing us to achieve considerably higher sequence depth and coverage than could be achieved through Sanger sequencing. The resulting high-volume NGS datasets make it possible to employ innovative new methodologies into our bioinformatics analyses of polyadenylation that will advance our understanding of APA and its regulatory mechanisms. Specifically, poly(A) sites will be more accurately annotated and cataloged based on their locations (e.g. within introns vs. exons), types (e.g. antisense vs. sense), and biological consequences (e.g. non-sense mediated decay, protein product truncation, and modification of micro-RNA binding sites). We will study how APA patterns and relevant cis-regulatory motifs have changed in different species, genotypes, tissues, developmental stages and environmental stresses. We will validate our predicted findings through wet lab protocols. Finally we will release our data, protocols and software to the research community through a richly analyzed and visualized online database and web service. Completion of the aims of this project for our model organisms will pave the way to more complete understanding of these complex regulatory mechanisms in Humans and other eukaryotes. PUBLIC HEALTH RELEVANCE: When eukaryotic genes are expressed, their mRNAs have to be processed during maturation, a step of which is polyadenylation: the attachment of a poly(A) tail to mark and protect the end of mRNA. However, alternative polyadenylation (APA) at a different poly(A) site of mRNA will result in information lose of the mRNA and has been linked to cause cancers and other diseases. Using Next-Generation Sequencing data, we will employ innovative methodologies to accurately annotate poly(A) sites, detect poly(A) signals, examine APA and its regulatory mechanisms, and provide biomedical/biological research communities with an information rich database that will advance our understanding of APA role in human diseases.