Cellular Surveillance and Degradation of Aberant tRNA

描述（由申请人提供）：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也在启动RNA指导的几种逆转录病毒基因组（包括HIV）的DNA合成中发挥作用。因此，阐明tRNA形成和功能的机制对基因表达的生理和病理形式具有意义。新合成的前体转运RNA经过广泛的加工，成为核质运输和蛋白质合成的能力。大多数tRNA具有相似的三级结构，这取决于多种分子内相互作用和许多修饰的核苷。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tRNA将是有益的。降解有缺陷的tRNA的机制已在E. coli中，但对从真核细胞中去除异常的tRNA一无所知。酵母tRNA 1-甲基腺苷（m1A）甲基转移酶是由TRM 6和TRM 61编码的双亚基酶。在trm 6和trm 61突变体中，m1A形成被阻断，tRNAi Met水平降低，表明m1A对tRNAi Met稳定性至关重要。研究不稳定性的机制和m1A在tRNA周转中的作用是该提案的重点。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（sup 1 -3），恢复突变体trm 6 -504中的tRNAi Met水平。从这些观察结果和这些基因中的两个的身份，我们已经建立了缺乏m1A的tRNAi Met被识别为腺苷酸化并被细胞核中的外泌体消除。将评估外泌体的所有亚基是否在降解低修饰的tRNAi Met中同等地起作用，并将测试外泌体的辅助组分的参与。我们的工作将集中在揭示这种tRNA降解机制的细节，通过分配生化活性的蛋白质确定为途径的组成部分。我们还将询问降解是否仅限于低修饰的tRNA，或者其他不稳定的tRNA或RNA是否易于在该途径中周转。这项研究将提供有关mIA在tRNA代谢中的重要性的见解，并提供了切实的证据，一种新的细胞途径，用于监测tRNA加工和消除真核细胞中的异常tRNA。

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.