tRNA Processing

摘要 TRNA在所有生物体中都高度进化，通过同源tRNA合成酶进行特异性识别， 保真度解码，翻译使用高效，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起着至关重要的作用 健康。TRNA小体(反密码子环外)的修饰对于tRNA在酵母中的稳定性至关重要，并且 与人类的几种神经疾病有关。我们研究了苏云金杆菌快速tRNA衰变(RTD)途径。 Cerevisiae，其靶向成熟tRNA的子集，由于暴露而缺乏任何一种身体修饰 5‘-3’核酸外切酶Rat1和Xrn1的5‘端。RTD也经常出现在tRNA变种中， 破坏稳定的突变暴露在5‘端，并在met22Δ突变体中被抑制，因为增加了 3‘，5’二磷酸腺苷(Pap)及其对Rat1和Xrn1的抑制作用 人们对RTD或任何其他真核生物的身体修饰生物学知之甚少。为了解决这个问题，我们 正在研究裂解酵母中的这些过程，因为它的~6亿 与酿酒酵母的进化距离很远，这是因为它的遗传学和分子生物学很简单。 我们最近在酿酒酵母中发现了一种不寻常的衰退途径，在这种途径中，前tRNA被降解。 在细胞质中通过一条由Met22调控的途径。这种由Met22调节的前tRNA衰退(MPD)途径是 独立于RTD，因为与经典的RTD不同，它不需要Rat1或Xrn1核酸外切酶和 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和核外切体作用于细胞核中的前tRNAs。更确切地说， MPD发生在未剪接的Pre-tRNA上，由于内含子-外显子结构受损，它聚集在细胞质中。 我们还研究了反密码子环中的修饰，因为它们在翻译中的重要性，重点是 Trm7，它2‘-O-甲基化某些tRNA的反密码子环中的N32和N34。酿酒葡萄球菌和庞氏葡萄球菌 Trm7突变有严重的生长缺陷，而突变的人类则有智力残疾。我们的前辈 结果表明，酿酒酵母和金黄色葡萄球菌trm7突变体的生长缺陷是由于生长缺陷减少所致。 TRNAPhe的功能，但不是减少量。我们最近发现了一种不同寻常的酿酒酵母特性 和S.pombe trm7Δ突变体：每个突变体都有力地激活了总氨基酸控制(Gaac) 响应，它大规模地重新编程所有真核生物中的基因表达，这是由于不带电荷的tRNA被 GCN2K，但trm7tRNA突变体不表现出可检测到的Δ电荷缺陷。 作为后续研究，我们将：1)研究RTD途径和机体修饰的异同 在S.pombe中的生物学定义了Met22调节的反密码子茎变体的前tRNA衰退是如何在S.pombe中发生的。 3)定义了trm7Δ突变体如何激活Gaac途径，以及trm7如何识别tRNA。

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.