The role of polyadenylation in RNA turnover

描述(由申请人提供)： 项目摘要本项目的长期目标仍然是了解多聚腺苷在原核生物转录后调控中所起的作用。虽然长期以来被认为是真核生物特有的特征，但在过去的10年里，研究表明，多聚腺苷酸化与大肠杆菌中的mRNA衰退、rRNA加工、tRNA成熟和整体RNA质量控制密切相关。同样重要的是，现在已经证明，在几乎所有原核生物中，通过多聚(A)聚合酶或多核苷酸磷酸化酶进行的多聚腺苷参与了RNA的转录后修饰。此外，在过去的几年中，已经证明真核生物包含一个核聚腺苷酸化系统，该系统非常类似于大肠杆菌系统，因为它针对有缺陷的Pol II转录本进行降解。因此，大肠杆菌是研究这一复杂系统的一个很好的原核模型系统。基于我们在当前赠款期间所取得的进展，我们现在能够对多聚腺苷化的生化机制及其在mRNA衰退、tRNA成熟和rRNA加工中所起的作用有更深入的了解。具体的实验方法包括：1.分析体内和体外多聚腺苷化复合体的组成和功能；2.确定Rho非依赖性转录终止子作为多腺化信号的基本要求；3.确定PAP I优先使全长或部分降解的转录本多腺化；4.确定多腺化在rRNA和tRNAs成熟中的作用；以及5.分析Poly(A)尾巴与以RNase E为基础的降解体之间在启动mRNA衰变中的相互作用。由于多聚腺苷似乎存在于包括许多病原体在内的广泛的原核生物中，因此对这一系统的更完整的了解为确定新的抗微生物靶标提供了机会，这些靶标将对革兰氏阴性或革兰氏阳性细菌具有活性。公共卫生相关性：项目叙述虽然长期以来，多聚腺苷酸化一直被认为是真核生物的一个独特特征，但在过去的十年里，它已被证明与模式生物大肠杆菌中的mRNA衰退、rRNA加工和tRNA成熟密切相关。同样重要的是，现在已经证明，在几乎所有的原核生物中，多聚腺苷参与了RNA的转录后修饰。因此，本申请中提出的实验旨在通过结合生物化学、遗传学和生物信息学的实验方法，更深入地了解聚腺苷酸化的生化机制以及它在mRNA衰退、rRNA加工和tRNA成熟中所扮演的角色。

DESCRIPTION (provided by applicant): Project Summary The long range goal of this project still remains to understand the role that polyadenylation plays in post-transcriptional regulation in prokaryotes. Although long considered to be a feature unique to eukaryotes, over the past 10 years it has been shown that polyadenylation is intimately involved in mRNA decay, rRNA processing, tRNA maturation and overall RNA quality contol in Escherichia coli. Of equal importance, it has now been shown that polyadenylation, either by poly(A) polymerase type enzymes or polynucleotide phosphorylase are involved in the post-transcriptional modification of RNA in almost all prokaryotes. In addition, over the past few years it has been shown that eukaryotes contain a nuclear polyadenylation system that closely mimics the E. coli system in that it targets defective Pol II transcripts for degradation. Thus E. coli is an excellent prokaryotic model system for studying this complex system. Based on the progress that we have made during the current grant period, we are now in a position to develop a much more sophisticated understanding of both the biochemical mechanism of polyadenylation and the roles it plays in mRNA decay, tRNA maturation and rRNA processing. Specific experimental approaches include: 1. Analyze the composition and function of the polyadenylation complex in vivo and in vitro; 2. Determine the essential requirements for a Rho-independent transcription terminator to function as a polyadenylation signal; 3. Determine whether PAP I preferentially polyadenylates full-length or partially degraded transcripts; 4. Determine the role of polyadenylation in the maturation of rRNAs and tRNAs; and 5. Analyze the interactions between poly(A) tails and the RNase E-based degradosome in the initiation of mRNA decay. Since polyadenylation appears to be present in a wide range of prokaryotes, including many pathogenic organisms, a more complete understanding of this system provides an opportunity to identify new targets for anti-microbials that will be active against either gram negative or gram positive bacteria. PUBLIC HEALTH RELEVANCE: Project Narrative Although polyadenylation has long been considered to be a unique feature of eukaryotes, over the past ten years it has been shown to be intimately involved in mRNA decay, rRNA processing and tRNA maturation in the model organism Escherichia coli. Of equal importance, it has now been demonstrated that polyadenylation is involved in the post-transcriptional modification of RNAs in almost all prokaryotes. Thus the experiments proposed in this application are designed to develop a much more sophisticated understanding of both the biochemical mechanism of polyadenylation and the roles that it plays in mRNA decay, rRNA processing and tRNA maturation through a combination of biochemical, genetic, and bioinformatic experimental approaches.