tRNA Processing

描述（由申请人提供）： 摘要tRNA的结构、序列和广泛的修饰使其被合成酶特异性识别，并在翻译过程中被统一使用。tRNA修饰在物种之间高度保守，并且在细胞功能和人类健康中具有许多作用。然而，我们对修饰的确切作用的理解在酵母中一直是难以捉摸的，正如这里所研究的那样，在人类中。 该项目的一个长期目标是确定一个tRNA质量控制途径，以监测成熟tRNA的完整性。该实验室先前表明，酵母trm8-trm4-突变体（缺乏7-甲基鸟苷和5-甲基胞苷）对温度敏感，这是由于成熟tRNAVal（AAC）通过未知途径的快速tRNA衰变（RTD）。最近的研究表明，RTD由5'-3 '核酸外切酶Rat 1和Xrn 1以及Met 22介导，并降解缺乏不同修饰的菌株中的不同特异性成熟tRNA种类，但不降解缺乏相同修饰的其他tRNA种类。随后的分析表明，特定的tRNA是RTD的目标，因为它们的受体和T茎不太稳定，并导致其5 '端暴露增加，修饰通过对三级结构的影响间接影响RTD，并且Xrn1在体外选择性降解RTD底物tRNA。此外，初步结果暗示RTD中的翻译机制的组件，因为RTD受到延伸因子EF-1A的影响，其通常结合带电的tRNA以递送到核糖体，并且受到Bud27的影响，其影响翻译起始，并且据报道结合EF-1A。 第二个长期目标是了解反密码子环周围修饰的生物学，以阐明它们对翻译的影响。目前的工作集中在C32（Cm32）和N34的2'-O-甲基化，这发生在三种tRNA上，需要Trm7，以及C32（m3C32）的3-甲基胞苷修饰，这发生在其他六种tRNA上，尽管还不清楚这些tRNA如何区分每种修饰。最近的研究结果表明，肌动蛋白结合蛋白Abp140（Trm140）的结构域是形成m3C32所需的六种tRNA的每一种，和trm140突变体是适度的翻译缺陷。初步研究还表明，严重的trm7生长缺陷是由于未能修饰C32处的一种特定tRNA引起的，并暗示了Trm7活性中先前未被识别的第二亚基。这可能对人类健康有影响，因为缺乏Trm7与智力迟钝有关。 为了落实这些成果，提出了四个目标：（1）确定RTD途径识别和降解tRNA底物的机制（2）确定Trm7和Trm140催化的反密码子环修饰的作用（3）确定m3C32、Cm32、和Nm34修饰;（4）利用基因组学方法探讨tRNA功能的限制、RTD底物和条件突变体。 公共卫生相关性： 细胞中所有蛋白质的合成都需要在核糖体翻译过程中通过转运RNA（tRNA）解码信使RNA。tRNA是高度修饰的，并且这些修饰在包括人类在内的所有生物体中是高度保守的;此外，许多人类健康状况和疾病与tRNA修饰的缺陷有关。该项目旨在研究真核生物酿酒酵母中tRNA修饰的作用，其中可以非常详细地研究生物学功能，以便随后可能应用于人类条件。

DESCRIPTION (provided by applicant): ABSTRACT The structure, sequence, and extensive modifications of tRNAs make them remarkably well designed for specific recognition by synthetases, and uniform use during translation. tRNA modifications are highly conserved among species, and have a number of roles in cell function and human health. However, our understanding of the precise roles of modifications has been elusive in yeast, as studied here, and in humans. One long term goal of this project is to define a tRNA quality control pathway that monitors the integrity of mature tRNA. The lab previously showed that yeast trm8- trm4- mutants (which lack 7-methylguanosine and 5-methylcytidine) are temperature sensitive due to rapid tRNA decay (RTD) of mature tRNAVal(AAC) by an unknown pathway. Recent work shows that RTD is mediated by the 5'-3' exonucleases Rat1 and Xrn1, and by Met22, and degrades different specific mature tRNA species in strains lacking different modifications, but not other tRNA species lacking the same modifications. Subsequent analysis indicates that specific tRNAs are targeted for RTD because their acceptor and T-stems are less stable and cause increased exposure of their 5' end, that modifications impact RTD indirectly through their effect on tertiary structure, and that Xrn1 selectively degrades RTD substrate tRNAs in vitro. Moreover, preliminary results implicate components of the translation machinery in RTD, since RTD is affected by the elongation factor EF-1A, which normally binds charged tRNA for delivery to the ribosome, and by Bud27, which affects translation initiation, and is reported to bind EF-1A . A second long term goal is to understand the biology of modifications around the anticodon loop to clarify their effects on translation. Current work is focusing on 2'-O-methylation of C32 (Cm32) and N34, which occurs on three tRNAs and requires Trm7, and on 3-methylcytidine modification of C32 (m3C32), which occurs on six other tRNA species, although it is unclear how these tRNAs are distinguished for each modification. Recent results showed that a domain of the actin binding protein Abp140 (Trm140) is required for formation of m3C32 for each of the six tRNA species, and that trm140- mutants are modestly translation defective. Preliminary work also suggests that the severe trm7- growth defect is caused by failure to modify one particular tRNA at C32, and implicates a previously unrecognized second subunit in Trm7 activity. This may have implications in human health since lack of Trm7 is associated with mental retardation. To follow up on these results, four aims are proposed: (1) To determine the mechanism by which the RTD pathway recognizes and degrades tRNA substrates (2) To determine the roles of anticodon loop modifications catalyzed by Trm7 and Trm140 (3) To define substrate specificity for m3C32, Cm32, and Nm34 modifications by Trm7 and Trm140 and (4) To use genomic methods to probe limits of tRNA function, RTD substrates and conditional mutants. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Synthesis of all proteins in cells requires decoding of messenger RNA by transfer RNA (tRNA) during translation by the ribosome. tRNAs are highly modified, and these modifications are highly conserved in all organisms, including humans; moreover, a number of human health conditions and diseases are associated with defects in tRNA modifications. This project is directed toward study of the role of tRNA modifications in the model eukaryotic organism Saccharomyces cerevisiae, in which it is possible to study biological function in great detail, for possible subsequent application to human conditions.