tRNA Processing

tRNA加工

• 批准号: 9885747
• 负责人: Eric M. Phizicky
• 金额: $ 44.41万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9885747
• 关键词: 7-methylguanosine; Address; Adenosine; Amino Acids; Amino Acyl-tRNA Synthetases; Anticodon; Binding; Biology; Cell Nucleus; Charge; Coupled; Cytoplasm; Defect; Distant; Eukaryota; Exons; Exonuclease; Exposure to; Fission Yeast; Gene Expression; Genetic; Growth; Health; Human; Impairment; Intellectual functioning disability; Introns; Measures; Messenger RNA; Methods; Methylation; Mitochondrial Diseases; Modification; Molecular Biology; Monitor; Mutation; Nuclear; Organism; Orthologous Gene; Pathway interactions; Peptides; Phenylalanine-Specific tRNA; Phosphotransferases; Post-Translational Protein Processing; Process; Proline-Specific tRNA; Property; RNA Splicing; Ribosomes; Role; Saccharomyces cerevisiae; Source; Structure; Temperature; Testing; Transfer RNA; Transfer RNA Aminoacylation; Translations; Variant; X-linked mental retardation 9; Yeasts; exosome; follow-up; inorganic phosphate; mutant; nervous system disorder; novel; nuclease; prevent; response; restoration; spleen exonuclease; stem; tRNA Precursor; temperature sensitive mutant; transcriptome sequencing

ABSTRACT tRNAs are highly evolved in all organisms for specific recognition by cognate tRNA synthetases, high fidelity decoding, efficient use in translation, and high stability. The ubiquitous tRNA modifications are highly conserved in eukaryotes, and many have crucial roles in the yeast Saccharomyces cerevisiae and in human health. Modifications in the tRNA body (outside the anticodon loop) are crucial for tRNA stability in yeast, and associated with several neurological disorders in humans. We study the rapid tRNA decay (RTD) pathway in S. cerevisiae, which targets a subset of mature tRNAs lacking any of several body modifications, due to exposure of the 5' end to the 5'-3' exonucleases Rat1 and Xrn1. RTD also frequently occurs in tRNA variants with destabilizing mutations exposing the 5' end, and is inhibited in met22Δ mutants due to increased levels of adenosine 3',5' bis-phosphate (pAp) and its inhibition of Rat1 and Xrn1. Little is known about RTD or the biology of body modifications in any other eukaryote. To address this, we are studying these processes in the fission yeast Schizosaccharomyces pombe because of its ~600 million years evolutionary distance from S. cerevisiae, and because of its facile genetics and molecular biology. We have recently uncovered an unusual decay pathway in S. cerevisiae in which pre-tRNAs are degraded in the cytoplasm by a pathway regulated by Met22. This Met22-regulated pre-tRNA decay (MPD) pathway is independent of RTD, because unlike classical RTD, it does not require the Rat1 or Xrn1 exonucleases and does not act on mature tRNA, and it is novel because it is also independent of the nuclear surveillance tRNA decay pathway, which acts in the nucleus on pre-tRNAs through Trf4, RRP6 and the nuclear exosome. Rather, MPD occurs on unspliced pre-tRNA that accumulates in the cytoplasm due to impaired intron-exon structure. We also study modifications in the anticodon loop, due to their importance in translation, with a focus on Trm7, which 2’-O-methylates N32 and N34 in the anticodon loop of certain tRNAs. S. cerevisiae and S. pombe trm7 mutants have severe growth defects, while humans with mutations have intellectual disability. Our prior results showed that the growth defect of S. cerevisiae and S. pombe trm7 mutants was due to reduced function, but not reduced amounts, of tRNAPhe. We recently discovered an unusual property of S. cerevisiae and S. pombe trm7Δ mutants: each mutant robustly activates the general amino acid control (GAAC) response, which massively reprograms gene expression in all eukaryotes due to uncharged tRNA sensed by Gcn2 kinase, but trm7Δ mutants do not exhibit a detectable tRNA charging defect. To follow up, we will: 1) Examine similarities and differences in the RTD pathway and body modification biology in S. pombe 2) Define how Met22-regulated pre-tRNA decay of anticodon stem variants occurs in S. cerevisiae 3) Define how trm7Δ mutants activate the GAAC pathway and how Trm7 recognizes tRNAs.

抽象的 tRNA 在所有生物体中都高度进化，可被同源 tRNA 合成酶特异性识别，高 保真解码，翻译高效，稳定性高。普遍存在的 tRNA 修饰是高度 在真核生物中保守，许多在酿酒酵母和人类中具有至关重要的作用 健康。 tRNA 体（反密码子环之外）的修饰对于酵母中 tRNA 的稳定性至关重要，并且 与人类的多种神经系统疾病有关。我们研究了 S. tRNA 快速衰减 (RTD) 途径。 cerevisiae，其目标是成熟 tRNA 的子集，由于暴露而缺乏任何几种身体修饰 5'-3' 核酸外切酶 Rat1 和 Xrn1 的 5' 末端。 RTD 也经常发生在 tRNA 变体中 暴露 5' 末端的不稳定突变，并且由于 水平增加而在 met22Δ 突变体中受到抑制 腺苷 3',5' 二磷酸 (pAp) 及其对 Rat1 和 Xrn1 的抑制。 人们对 RTD 或任何其他真核生物身体修饰的生物学知之甚少。为了解决这个问题，我们 正在研究裂殖酵母裂殖酵母中的这些过程，因为它有约 6 亿 与酿酒酵母的进化距离长达数年，并且由于其简单的遗传学和分子生物学。 我们最近在酿酒酵母中发现了一种不寻常的降解途径，其中前 tRNA 被降解 通过 Met22 调节的途径进入细胞质。这种 Met22 调节的前 tRNA 衰变 (MPD) 途径是 独立于 RTD，因为与经典 RTD 不同，它不需要 Rat1 或 Xrn1 核酸外切酶，并且 不作用于成熟的 tRNA，它是新颖的，因为它也独立于核监视 tRNA 衰变途径，通过 Trf4、RRP6 和核外泌体在细胞核中作用于前 tRNA。相当， MPD 发生在未剪接的前 tRNA 上，由于内含子-外显子结构受损，该前 tRNA 积聚在细胞质中。 我们还研究反密码子环的修饰，因为它们在翻译中很重要，重点是 Trm7，对某些 tRNA 的反密码子环中的 N32 和 N34 进行 2’-O-甲基化。酿酒酵母和粟酒裂殖酵母 trm7突变体有严重的生长缺陷，而突变的人类则有智力障碍。我们之前的 结果表明，酿酒酵母和粟酒裂殖酵母trm7突变体的生长缺陷是由于 tRNAPhe 的功能，但数量并未减少。我们最近发现了酿酒酵母的一个不寻常的特性 和 S. pombe trm7Δ 突变体：每个突变体都会强烈激活一般氨基酸控制 (GAAC) 反应，由于不带电的 tRNA 感应到，它会大规模地重新编程所有真核生物中的基因表达 Gcn2 激酶，但 trm7Δ 突变体不表现出可检测的 tRNA 充电缺陷。 后续，我们将：1）检查 RTD 途径和身体改造的异同 粟酒裂殖酵母中的生物学 2) 定义在裂殖酵母中 Met22 调节的反密码子茎变体的前 tRNA 衰变是如何发生的 酿酒酵母 3) 定义 trm7Δ 突变体如何激活 GAAC 途径以及 Trm7 如何识别 tRNA。