RNA decay and processing activities of the RNA exosome

描述（由申请人提供）：几乎所有RNA都是通过各种酶（包括RNA酶）从初级转录物加工成成熟RNA的。在这个过程中会发生许多错误，以这种方式产生的异常RNA会迅速降解以维持RNA稳态。RNA外泌体在其中起着几个关键作用。首先，核外泌体处理一些来自较长前体的rna。例如，它处理来自300 nt前体的160 nt 5.8S rRNA。其次，核外泌体完全降解一些作为基因表达副产物的rna，包括作为rRNA前体一部分的5'外部转录间隔物。第三，核外泌体降解不能完成正常加工的异常rna。这包括未适当修改的启动器tRNA。第四，外泌体也存在于细胞质中，在那里它执行mRNA衰变的两种一般途径之一。第五，胞质外显体对于降解异常mrna尤其重要，包括那些缺乏停止密码子和缺乏聚(a)尾的mrna。可能与降解异常mrna的过程有关的是外泌体具有抗病毒活性。这项研究的长期目标是了解RNA外泌体在这些加工和衰变途径中的作用。核心外泌体由10个蛋白组成，其中Rrp44p是催化亚基。我们和其他人之前已经证明Rrp44p含有一个3‘到5’的外核分解结构域和一个核内分解结构域。我们建议研究以更好地了解内切酶活性对外泌体功能的贡献。虽然Rrp44p是核心外泌体的唯一催化亚基，但其他9个核心亚基对于生存能力和所有外泌体功能也至关重要。我们提出了旨在更好地理解其他九个亚单位的作用的实验。虽然纯化的外泌体在体外具有RNase活性，但其在体内的活性需要许多辅助因子。虽然其中一些辅助因子仅对外泌体的特定作用是必需的，但所有外泌体功能都需要ski2样家族的RNA解旋酶。外泌体的所有细胞质功能都需要Ski2p本身，而密切相关的Mtr4p则需要外泌体的所有核功能。我们提出的实验旨在阐明这些解旋酶如何协助外泌体发挥其许多功能。

DESCRIPTION (provided by applicant): Almost all RNAs are processed from a primary transcript to a mature RNA by a variety of enzymes, including RNases. During this process many mistakes are made and aberrant RNAs generated this way are rapidly degraded to maintain RNA homeostasis. The RNA exosome plays several critical roles in this. First, the nuclear exosome processes some RNAs from longer precursors. For example, it processes the 160 nt 5.8S rRNA from a 300 nt precursor. Second, the nuclear exosome completely degrades some RNAs that are byproducts of gene expression, including the 5' external transcribed spacer that is part of the rRNA precursor. Third, the nuclear exosome degrades aberrant RNAs that fail to complete proper processing. This includes the initiator tRNA that is not properly modified. Fourth, the exosome is also present in the cytoplasm, where it performs one of two general pathways of mRNA decay. Fifth, the cytoplasmic exosome appears especially important for degrading aberrant mRNAs, including those that lack a stop codon and those that lack a poly(A) tail. Probably related to the process of degrading aberrant mRNAs is that the exosome has an antiviral activity. The long term goal of this research is to understand the role of the RNA exosome in these processing and decay pathways. The core exosome consists of ten proteins, of which Rrp44p is the catalytic subunit. We and others have previously shown that Rrp44p contains a 3' to 5' exoribonucleolytic domain and an endonucleolytic domain. We propose research to better understand the contributions of the endonuclease activity to exosome function. While Rrp44p is the only catalytic subunit of the core exosome, the other nine core subunits are also essential for viability and for all exosome functions examined. We propose experiments aimed at better understanding the role of the other nine subunits. Although the purified exosome has RNase activity in vitro, its in vivo activity requires many cofactors. While some of these cofactors are required only for specific roles of the exosome, an RNA helicase of the Ski2-like family is required for all exosome functions. Ski2p itself is required for all cytoplasmic functions of the exosome, while the closely related Mtr4p is required for all nuclear functions of the exosome. Our proposed experiments seek to clarify how these helicases assist the exosome in its many function. PUBLIC HEALTH RELEVANCE: This research will use yeast as a model organism, but the exosome and its cofactors is highly conserved between yeast and humans. Thus the results should increase our understanding of the role of RNA processing and RNA degradation by the exosome in gene expression in humans as well.