Cellular Surveillance and Degradation of Aberant tRNA

描述(申请人提供)：转移RNA用于将信使RNA中的信息解释为进行细胞活动的蛋白质。值得注意的是，细胞tRNA还在启动RNA引导的几个逆转录病毒基因组的DNA合成方面发挥作用，包括艾滋病毒。因此，阐明tRNA的形成和功能机制对基因表达的生理和病理形式具有重要意义。新合成的Pre-tRNA经过广泛的加工，变得能够进行核质运输和蛋白质合成。大多数tRNA获得类似的三级结构，这取决于各种分子内相互作用和大量修饰的核苷。这种结构对tRNA的稳定性和功能至关重要。为了保持蛋白质合成的保真度，细胞检测和消除异常的tRNAs将是有益的。在大肠杆菌中已经描述了一种降解有缺陷的tRNAs的机制，但关于从真核细胞中去除异常tRNAs的情况尚不清楚。酵母tRNA1-甲基腺苷(M1a)甲基转移酶是由TRM6和TRM61编码的双亚单位酶。在trm6和trm61突变体中，M1a的形成被阻断，tRNAi Met水平降低，表明M1a对tRNAi Met的稳定性至关重要。研究不稳定的机制和M1a在tRNA周转中的作用是这一提议的重点。为了确定克服M1A缺陷的细胞策略，我们分离了三个抑制基因(sup1-3)，它们可以恢复突变体trm6-504中tRNAi Met的水平。根据这些观察和其中两个基因的鉴定，我们已经建立了缺乏M1a的tRNAi Met被细胞核中的外切体识别、腺化和消除。将评估外切体的所有亚单位是否在降解低修饰的tRNAi Met方面具有同等的功能，并将测试外切体的辅助成分的参与。我们的工作将集中在通过将生化活性分配给被识别为该途径的组成部分的蛋白质来揭示这种tRNA降解机制的细节。我们还会问，降解是否仅限于低修饰的tRNAs，或者其他不稳定的tRNAs或RNAs是否容易在这一途径中发生改变。这项研究将深入了解MLA在tRNA代谢中的重要性，并为监测tRNA加工和消除真核细胞中异常tRNA提供了新的细胞途径的确凿证据。

DESCRIPTION (provided by applicant): Transfer RNA serves to interpret the information in messenger RNA into proteins that conduct cellular activities. Remarkably, cellular tRNAs also play a role in priming RNA-directed DNA synthesis of several retroviral genomes, including HIV. Thus, elucidating the mechanisms of tRNA formation and function has implications for physiological and pathological forms of gene expression. Newly synthesized pre-tRNAs undergo extensive processing to become competent for nucleocytoplasmic transport and protein synthesis. Most tRNAs attain a similar tertiary structure that depends on a variety of intramolecular interactions and numerous modified nucleosides. This structure is critical for tRNA stability and function. To preserve the fidelity of protein synthesis, it would be beneficial for the cell to detect and eliminate aberrant tRNAs. A mechanism to degrade defective tRNAs has been described in E. coli, but nothing is known about the removal of aberrant tRNAs from eukaryotic cells. The yeast tRNA 1-methyladenosine (m1A) methyltransferase, is a two-subunit enzyme encoded by TRM6 and TRM61. In trm6 and trm61 mutants, m1A formation is blocked and tRNAi Met levels are diminished, suggesting that m1A is critical for tRNAi Met stability. Investigating the mechanism of instability and the role of m1A in tRNA turnover are the focus of this proposal. To identify cellular strategies for overcoming m1A deficiency, we isolated three suppressor genes (sup1-3) that restore tRNAi Met levels in the mutant trm6-504. From these observations and the identity of two of these genes, we have established that tRNAi Met lacking m1A is recognized adenylated and eliminated by the exosome in the nucleus. Whether all subunits of the exosome function equally in degrading hypomodified tRNAi Met will be assessed and the involvement of accessory components of the exosome will be tested. Our work will focus on uncovering the details of this tRNA degradation mechanism by assigning biochemical activities to proteins identified as integral components of the pathway. We will also ask if degradation is limited to hypomodified tRNAs or if other destabilized tRNAs or RNAs are susceptible to turnover in this pathway. This study will provide insight regarding the importance of mlA in tRNA metabolism and has provided tangible evidence of a novel cellular pathway for the surveillance oftRNA processing and the elimination of aberrant tRNAs from eukaryotic cells.