Cellular Surveillance and Degradation of Aberant tRNA

异常 tRNA 的细胞监视和降解

• 批准号: 6869314
• 负责人: JAMES T ANDERSON
• 金额: $ 20.54万
• 依托单位: MARQUETTE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6869314
• 关键词: DNA directed DNA polymerase; Saccharomyces cerevisiae; adenosine; chemical stability; enzyme activity; fungal genetics; fungal proteins; gene mutation; genetic regulation; high performance liquid chromatography; immunoprecipitation; nucleic acid metabolism; nucleic acid sequence; posttranscriptional RNA processing; site directed mutagenesis; small nuclear RNA; transfer RNA

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.

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 还在几种逆转录病毒基因组（包括 HIV）的 RNA 指导的 DNA 合成中发挥着作用。因此，阐明 tRNA 形成和功能的机制对于基因表达的生理和病理形式具有重要意义。新合成的前 tRNA 经过广泛的加工，能够进行核质运输和蛋白质合成。大多数 tRNA 都具有类似的三级结构，该结构取决于各种分子内相互作用和大量修饰的核苷。这种结构对于 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，细胞检测并消除异常的 tRNA 将是有益的。大肠杆菌中已经描述了一种降解缺陷 tRNA 的机制，但对于如何从真核细胞中去除异常 tRNA 却一无所知。酵母 tRNA 1-甲基腺苷 (m1A) 甲基转移酶是由 TRM6 和 TRM61 编码的双亚基酶。在 trm6 和 trm61 突变体中，m1A 形成被阻断，tRNAi Met 水平降低，表明 m1A 对于 tRNAi Met 稳定性至关重要。研究不稳定的机制以及 m1A 在 tRNA 周转中的作用是本提案的重点。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 (sup1-3)，它们可以恢复突变体 trm6-504 中的 tRNAi Met 水平。根据这些观察结果和其中两个基因的身份，我们确定缺乏 m1A 的 tRNAi Met 被细胞核中的外泌体识别并消除。将评估外泌体的所有亚基在降解低修饰 tRNAi Met 方面是否具有同等功能，并测试外泌体辅助成分的参与情况。我们的工作将侧重于通过将生化活性分配给被确定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。我们还将询问降解是否仅限于修饰不足的 tRNA，或者其他不稳定的 tRNA 或 RNA 是否容易受到该途径的周转影响。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 的新细胞途径提供了切实证据。