Regulation of eukaryotic mRNA polyadenylation by sustained stress

 描述(申请人提供)：真核细胞经常需要修改它们的基因表达模式，以适应环境变化或对调节细胞功能的信号做出反应。调节表达的一种快速方法是改变mRNAs的长度。3‘端的长度由多聚腺苷酸化决定，这是一个重要的加工步骤，涉及在Polya(Pa)位点切割mRNA前体，然后在新的3’端添加腺苷。改变Pa位点可以改变编码序列的数量或移除控制定位、翻译和稳定性的3‘非翻译区(UTR)中的重要调控序列。 大多数真核基因都含有两个或两个以上的PA位点，它们提供不同长度的mRNA异构体。在发育、分化和肿瘤形成过程中，这些异构体的比例发生了惊人的大量变化，并对细胞的环境做出了反应。这些发现表明，选择性多聚腺基化(APA)结合了转录启动和选择性剪接，是调节特定细胞状态所需mRNAs数量和类型的一种重要但未被认识的方式。然而，导致APA的机制还没有很好地确定，对蛋白质输出和对细胞功能的贡献的后果仍然知之甚少。我们的总体目标是了解PA网站使用的这些广泛变化的机制和功能后果。该领域以前的工作主要集中在3‘非编码区的变化，以及由于调控序列的获得或丢失而导致的转录后调控的变化。相反，尽管有明确的证据表明它们的普遍存在以及它们对细胞状态变化的系统变化，但相对较少的关注结束于编码序列内的Pa位点的转录本(CDS-Pa)。因此，我们将重点研究CDS-PA转录本。我们将检验这样一种假设，即这些异构体的平衡变化是由mRNA稳定性、mRNA聚腺苷酸化和转录终止的协调组合控制的，并且无论机制如何，这些变化都将影响蛋白质的产生，因此是对环境变化的适当反应的一个组成部分。我们的方法是创新的，因为它将使用全基因组表达分析来指导有重点的分子生物学实验，这些实验将探索基因表达调控重要步骤的潜在机制。这一建议意义重大，因为APA的广泛使用及其迅速影响细胞制造的蛋白质的数量和类型的潜力。这些目标的实现有望产生新的见解，广泛适用于由替代多聚腺苷调节的其他细胞状态。

 DESCRIPTION (provided by applicant): Eukaryotic cells often need to modify their gene expression patterns to adapt to environmental changes or to respond to signals regulating cell function. One rapid means of regulating expression is to vary the length of mRNAs. Length at the 3' end is determined by polyadenylation, an essential processing step involving cleavage of mRNA precursor at the polyA (pA) site, followed by addition of adenosines to the new 3' end. Changing the pA site can alter the amount of coding sequence or remove important regulatory sequences in the 3' untranslated region (UTR) that govern localization, translation, and stability. The majority of eukaryotic genes contain two or more pA sites which give mRNA isoforms of different length. Changes in the proportion of these isoforms occurs for a surprisingly large number of genes during development, differentiation, and tumorigenesis and in response to the cell's environment. These findings indicate that alternative polyadenylation (APA) joins transcription initiation and alternative splicing as an important, but under-appreciated way to modulate the amount and types of mRNAs needed for specific cellular states. However, the mechanisms leading to APA are not well defined, and the consequences on protein output and contribution to cell function remain poorly understood. Our overall objective is to understand the mechanism and functional consequences of these widespread changes in pA site usage. Previous work in the field has largely focused on changes to the 3'UTR, and the consequent changes in post-transcriptional regulation due to gain or loss of regulatory sequences. In contrast, relatively little attention has been given to transcripts ending at pA sites within the coding sequence (CDS-pA), despite clear evidence of their prevalence and their systematic variation in response to changes in cell state. We will therefore focus our studies on the CDS-pA transcripts. We will test the hypothesis that variations in the balance of these isoforms are controlled by a coordinated combination of changes in mRNA stability, mRNA polyadenylation, and transcription termination and that regardless of the mechanism, these changes will affect protein production and therefore are an integral component of an appropriate response to environmental changes. Our approach is innovative because it will use whole genome expression analysis to guide focused molecular biological experiments that will probe mechanisms underlying an important step in regulation of gene expression. This proposal is significant because of the wide-spread use of APA and its potential to rapidly affect the amount and type of protein made by a cell. Accomplishment of these aims is expected to yield novel insights broadly applicable to other cellular states modulated by alternative polyadenylation.