RNA decay and processing activities of the RNA exosome

描述（由申请人提供）：几乎所有的RNA都是通过多种酶（包括RNA酶）从初级转录物加工成成熟RNA的。在这个过程中，会产生许多错误，并且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，核外泌体从较长的前体加工一些RNA。例如，它从300 nt前体加工160 nt 5.8S rRNA。第二，核外泌体完全降解作为基因表达的副产物的一些RNA，包括作为rRNA前体的一部分的5'外部转录间隔区。第三，核外泌体降解不能完成正确加工的异常RNA。这包括未被适当修饰的起始tRNA。第四，外泌体也存在于细胞质中，在那里它执行mRNA衰变的两个一般途径之一。第五，细胞质外泌体对于降解异常mRNA尤其重要，包括那些缺少终止密码子和缺少poly（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种蛋白质组成，其中Rrp44 p是催化亚基。我们和其他人以前已经表明，Rrp44 p含有一个3'至5'核糖核酸外切结构域和一个核糖核酸内切结构域。我们建议进行研究，以更好地了解核酸内切酶活性对外泌体功能的贡献。 虽然Rrp44 p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力和所有外泌体功能也是必不可少的。我们提出的实验旨在更好地了解其他九个亚基的作用。 虽然纯化的外泌体在体外具有RNA酶活性，但其体内活性需要许多辅因子。虽然这些辅因子中的一些仅为外泌体的特定作用所需，但Ski2样家族的RNA解旋酶为所有外泌体功能所需。Ski2p本身是外泌体的所有细胞质功能所必需的，而密切相关的Mtr4p是外泌体的所有核功能所必需的。我们提出的实验试图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.