tRNA Processing

描述（由申请人提供）：tRNA因其在翻译中的作用而被精心设计，因为它们的整体结构足够均匀以供翻译装置可比使用，但又足够不同以用于翻译期间的特异性氨酰化和特异性解码。tRNA是迄今为止在细胞中发现的最常见的RNA种类，其成熟需要广泛的加工和大量的修饰。在酿酒酵母（Saccharomyces cerevisiae）中，平均胞质tRNA具有13个修饰残基，并且在不同的tRNA中总共存在25种不同的化学修饰。这些tRNA修饰在包括人类在内的不同生物体中高度保守。然而，理解许多修饰的作用一直是难以捉摸的，特别是对于远离反密码子的大量修饰，因为相应的突变体没有明显的表型。该项目的长期目标是确定酵母中发现的tRNA修饰的作用。该实验室已经证明，在其tRNA中缺乏m7 G的酵母突变体（trm 8或trm 82突变体）和缺乏远离反密码子的七种其他修饰中的任何一种，在高温下具有严重的生长缺陷，而缺乏这些修饰的其他组合的细胞没有缺陷。特别是，缺乏m7 G和m5 C的trm 8 trm 4突变体在升高的温度下死亡，因为它们的tRNAVal（AAC）被先前未描述的作用于成熟tRNA的质量控制途径快速降解。这是第一次描述去除细胞中成熟tRNA的途径，并且该途径不同于先前描述的唯一其他tRNA质量控制途径，该途径通过poly（A）聚合酶Trf 4和核外泌体作用于细胞核中的前tRNA。该实验室还研究了在几乎所有生物体中tRNAHis的5'端的-1位置（G-1）处发现的独特的额外鸟嘌呤核苷酸残基的形成，该位置在绝大多数tRNA中通常是未被占据的。鉴定了将G-1添加到tRNAHis的必需tRNAHis鸟苷酰转移酶（Thg 1），显示通过其反密码子识别tRNAHis，并显示在本实验室中催化不寻常的反向3 '-5'聚合酶活性，并被其他人牵连到细胞周期进程中。这项建议有四个广泛的目标。(1)鉴定trm 8-trm 4突变体中tRNAVal（AAC）降解途径的组成部分。（2）确定tRNAVal（AAC）降解的机制。(3)研究Thg 1在细胞中的重要作用和G-1对tRNAHis功能的重要性;（4）鉴定其他修饰的作用。最近，几种修饰和tRNA加工酶已涉及影响人类健康的条件。计划中的研究将有助于阐明人类的这些过程。计划叙述细胞中所有蛋白质的合成都需要核糖体中的转运RNA（transfer RNA，tRNA）对信使RNA进行解码。最近的研究表明，许多人类健康状况和疾病与加工tRNA并催化形成对其在细胞中的功能至关重要的tRNA修饰的酶的缺陷有关。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tRNA加工和修饰，以便随后应用于人类条件。

DESCRIPTION (provided by applicant): tRNAs are exquisitely designed for their role in translation, since their overall structures are uniform enough for comparable use by the translation apparatus, yet different enough for specific aminoacylation and specific decoding during translation. tRNA is by far the most common RNA species found in cells, and its maturation requires extensive processing and a large number of modifications. In the yeast Saccharomyces cerevisiae, the average cytoplasmic tRNA bears 13 modified residues, and there are a total of 25 different chemical modifications in different tRNAs. These tRNA modifications are very highly conserved in different organisms, including humans. However, understanding the roles of many modifications has been elusive, particularly for the large number that are remote from the anticodon, because the corresponding mutants have no obvious phenotype. The long term goal of this project is to define the roles of tRNA modifications found in yeast. This laboratory has shown that yeast mutants lacking m7G in their tRNA (trm8 or trm82 mutants) and lacking any one of seven other modifications remote from the anticodon, have severe growth defects at elevated temperature, whereas cells that lack other combinations of these modifications have no defect. In particular, trm8 trm4 mutants, which lack m7G and m5C, die at elevated temperature because their tRNAVal(AAC) is rapidly degraded by a previously undescribed quality control pathway that acts on mature tRNA. This is the first description of a pathway that removes mature tRNA in the cell, and this pathway differs from the only other previously described tRNA quality control pathway, which acts instead on pre-tRNA in the nucleus through the poly(A) polymerase Trf4 and the nuclear exosome. This laboratory has also examined formation of the unique extra guanine nucleotide residue found at the -1 position (G-1) of the 5' end of tRNAHis in virtually all organisms, a position that is normally unoccupied in the vast majority of tRNAs. The essential tRNAHis guanylyltransferase (Thg1) that adds G-1 to tRNAHis was identified, shown to recognize tRNAHis through its anticodon, and shown to catalyze an unusual reverse 3'-5' polymerase activity in this laboratory, and implicated by others in cell cycle progression. This proposal has four broad aims. (1) To identify the components of the tRNAVal(AAC) degradation pathway in trm8 trm4 mutants (2) To determine the mechanism of tRNAVal(AAC) degradation. (3) To examine the essential roles of Thg1 in the cell and the importance of G-1 for tRNAHis function, and (4) To identify the roles of other modifications. Recently, several modifications and tRNA processing enzymes have been implicated in conditions that affect human health. The proposed studies should cast light on these processes in humans.PROJECT NARRATIVE Synthesis of all proteins in cells requires decoding of the messenger RNA by transfer RNA (tRNA) in the ribosome. Recent studies have shown that a number of human health conditions and diseases are associated with defects in enzymes that process tRNA and catalyze the formation of tRNA modifications that are important for its function in the cell. 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 tRNA processing and modifications in great detail, for subsequent application to human conditions.