tRNA Processing

 描述（由申请人提供） tRNA的序列、结构和修饰都经过精心调整，以实现高保真的充电和解码，有效地用于翻译，并具有高稳定性。该项目的一个主要目标是确定酵母中主要质量控制途径的范围和特异性 酿酒酵母靶向成熟tRNA，缺乏快速tRNA衰变（RTD）的某些修饰，导致生长缺陷。我们之前发现RTD作用于缺乏一种或多种修饰的tRNA，由5 '-3'核酸外切酶Rat 1和Xrn 1介导，受met 22 Δ突变抑制，并且仅作用于缺乏特定修饰的tRNA种类的子集。理解这一途径的特异性是重要的，因为该领域的最新结果表明，RTD在HeLa细胞中是保守的，并且在某些小鼠修饰突变体和许多与tRNA突变相关的线粒体疾病中发生tRNA水平降低。先前对tRNASer家族的研究表明，RTD发生在具有不稳定的受体和T茎的完全修饰和低修饰的tRNA上，导致5'端暴露增加，与5'-3 '核酸外切酶的降解一致。然而，我们最近对tRNATyr功能的高通量分析（使用SUP 4 oc无义抑制子）提供了RTD也由不稳定的反密码子茎环（ASL）触发的证据，这表明了一种新的参与RTD的机制。此外，我们发现了一个意想不到的tRNA衰变途径的证据，发生在成熟的tRNA在RTD的情况下。 第二个主要项目集中在与酵母生长缺陷和人类疾病相关的选定修饰的生物学。几种不同的人类修饰基因中的任何一种缺陷都与智力残疾、小头畸形或家族性自主神经功能障碍有关。目前的一个主要目标是了解缺乏Trm 7的细胞的严重生长缺陷，Trm 7是3种tRNA物质的C32和N34的2 '-O-甲基化所必需的。人类TRM 7同源物FTSJ 1与非综合征性X连锁智力残疾（NSXLID）有关。我们发现S.酿酒酵母trm 7 Δ生长缺陷是由于缺乏功能性tRNAPhe，并且tRNAPhe经历了一系列复杂的修饰，其中Trm 7与Trm 732和Trm 734一起催化Cm 32和Gm 34修饰，然后促进m1 G37处的间布胞嘧啶（yW）形成。我们还发现，这种电路是保留和重要的远亲酵母裂殖酵母粟酒裂殖酵母。 接下来，我们提出：（1）明确tRNA在酵母中的识别和降解机制。(2)明确RTD通路的调控机制;（3）确定与人类疾病相关的选定修饰基因的作用，重点分析FTSJ 1病变的NSXLID患者细胞系，以及酵母Trm 7及其伴侣蛋白的作用和特异性。

 DESCRIPTION (provided by applicant) The sequence, structure, and modifications of tRNAs are exquisitely tuned for high fidelity charging and decoding, for efficient use in translation, and for high stability. One major goal of this project is to define the scope and specificity of a major quality control pathway in the yeast Saccharomyces cerevisiae that targets mature tRNAs lacking certain modifications for rapid tRNA decay (RTD), resulting in growth defects. We previously found that RTD acts on tRNAs lacking one or more of several modifications, is mediated by the 5'-3' exonucleases Rat1 and Xrn1, is inhibited by a met22Δ mutation, and acts on only a subset of tRNA species lacking the particular modifications. Understanding the specificity of this pathway is important because recent results in the field show that RTD is conserved in HeLa cells and that reduced tRNA levels occur in certain mouse modification mutants and in a number of mitochondrial diseases associated with tRNA mutations. Prior investigation of the tRNASer family showed that RTD occurs on fully modified and hypomodified tRNAs with destabilized acceptor and T-stems, resulting in increased exposure of the 5' ends, consistent with degradation by 5'-3' exonucleases. However, our recent high throughput analysis of tRNATyr function (using the SUP4oc nonsense suppressor) provides evidence that RTD is also triggered by a destabilized anticodon stem-loop (ASL), suggesting a new mechanism to engage RTD. In addition, we find evidence for an unexpected tRNA decay pathway that occurs on mature tRNAs in the absence of RTD. A second major project focuses on the biology of selected modifications associated with growth defects in yeast and with human disease. Defects in any of several different human modification genes are associated with intellectual disability, microcephaly, or familial dysautonomia. A major current goal is to understand the severe growth defect of cells lacking Trm7, which is required for 2'-O-methyation of C32 and N34 of 3 tRNA species. The human TRM7 homolog FTSJ1 is linked to non-syndromic X-linked intellectual disability (NSXLID). We found that the S. cerevisiae trm7Δ growth defect is due to lack of functional tRNAPhe and that tRNAPhe undergoes an intricate set of modifications in which Trm7 works with Trm732 and with Trm734 to catalyze Cm32 and Gm34 modifications, which then promote wybutosine (yW) formation at m1G37. We also found that this circuitry is retained and important in the distantly related yeast Schizosaccharomyces pombe. To follow up, we propose: (1) To define the mechanisms by which tRNA is recognized and degraded in yeast. (2) To define the mechanisms by which the RTD pathway is regulated, and (3) To determine the roles of selected modification genes implicated in human disease, with a focus on analysis of cell lines from NSXLID patients with FTSJ1 lesions, and on the roles and specificity of yeast Trm7 and its partner proteins.