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进一步了解真核生物中的替代性多聚腺苷酸化（阿帕），包括其发生的程度和调节机制。真核mRNA的成熟需要几个转录后过程的作用，其中之一是3 '端形成。该过程由前体mRNA切割和聚腺苷酸化组成，其中聚腺苷酸尾被添加到前体mRNA的新切割的3 '端。它由前体mRNA上的poly（A）信号基序引导。多聚腺苷酸化已经显示影响mRNA的稳定性、可翻译性和从细胞核到细胞质的可转运性。更复杂的是，许多真核生物基因都有不止一个poly（A）位点。选择性多聚腺苷酸化（阿帕）是利用这些不同的多聚腺苷酸位点产生具有相同基因的不同3 '端的mRNA转录物的现象。越来越多的证据表明，阿帕是调节基因表达的关键因素，影响mRNA水平和/或编码蛋白的功能。它还影响具有潜在顺式调节基序的3 '-UTR的性质和长度，所述顺式调节基序对于mRNA稳定性和翻译抑制是重要的。阿帕似乎受到发育和环境因素的调节，并且通常以组织和/或疾病特异性的方式发生。多聚腺苷酸信号或多聚腺苷酸化蛋白因子的突变导致包括癌症在内的严重疾病。然而，关于阿帕的潜在机制还有许多重要的问题没有回答。例如，目前还不清楚发育和环境线索如何转导到阿帕过程，或者什么特殊的poly（A）信号（即，需要特殊的RNA结构）来引导阿帕。下一代测序（NGS）彻底改变了转录组测序，使我们能够实现比桑格测序更高的序列深度和覆盖率。由此产生的大容量NGS数据集使我们有可能采用创新的新方法来进行多聚腺苷酸化的生物信息学分析，这将促进我们对阿帕及其调控机制的理解。具体而言，多聚腺苷酸位点将基于其位置（例如，内含子内与外显子内）、类型（例如，反义与有义）和生物学后果（例如，无义介导的衰变、蛋白质产物截短和微RNA结合位点的修饰）进行更准确的注释和编目。我们将研究阿帕模式和相关的顺式调控基序如何在不同物种、基因型、组织、发育阶段和环境胁迫下发生变化。我们将通过湿实验室协议验证我们的预测结果。最后，我们将通过丰富的分析和可视化的在线数据库和Web服务向研究社区发布我们的数据，协议和软件。完成本项目的目标，我们的模式生物将铺平道路，更完整地了解这些复杂的调控机制，在人类和其他真核生物。 公共卫生相关性：当真核基因表达时，它们的mRNA必须在成熟过程中被加工，其中一个步骤是聚腺苷酸化：连接聚腺苷酸尾以标记和保护mRNA的末端。然而，在mRNA的不同poly（A）位点上的选择性多聚腺苷酸化（阿帕）会导致mRNA的信息丢失，并与导致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poly（A）位点，检测poly（A）信号，检查阿帕及其调控机制，并为生物医学/生物学研究社区提供信息丰富的数据库，这将促进我们对阿帕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.