tRNA Processing

摘要 tRNA在所有生物体中高度进化，以通过同源tRNA合成酶进行特异性识别， 解码保真度高，翻译效率高，稳定性好。普遍存在的tRNA修饰高度 在真核生物中保守，许多在酵母酿酒酵母和人类中发挥关键作用 健康tRNA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， 与人类的几种神经系统疾病有关。研究了S. 酿酒酵母，其靶向缺乏几种身体修饰中的任何一种的成熟tRNA的子集， Rat 1和Xrn 1的5'端。RTD也经常发生在tRNA变体中， 在met 22 Δ突变体中， 腺苷3 '，5'二磷酸（pAp）及其对Rat 1和Xrn 1的抑制。 关于RTD或任何其他真核生物中的身体修饰生物学知之甚少。为了解决这个问题，我们 正在研究裂殖酵母裂殖酵母中的这些过程，因为它有大约6亿个 与S.酿酒酵母，并由于其简单的遗传学和分子生物学。 我们最近发现了一个不寻常的衰变途径在S。前体tRNA被降解的酿酒酵母 在细胞质中通过由Met 22调节的途径。这种Met 22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Rat 1或Xrn 1核酸外切酶， 不作用于成熟的tRNA，它是新颖的，因为它也不依赖于核监视tRNA 衰变途径，其在细胞核中通过Trf 4、RRP 6和核外泌体作用于pre-tRNA。相反地， MPD发生在由于内含子-外显子结构受损而在细胞质中积累的未剪接的前tRNA上。 我们还研究了反密码子环中的修饰，由于它们在翻译中的重要性，重点是 Trm 7，其2 '-O-甲基化某些tRNA的反密码子环中的N32和N34。S. cerevisiae和S. pombe trm 7突变体具有严重的生长缺陷，而具有突变的人具有智力残疾。我们事先 结果表明，S. cerevisiae和S. pombe trm 7突变体是由于减少 功能，但不是减少量，tRNAPhe。我们最近发现了S的一个不寻常的性质。酿酒酵母 和S.粟酒裂殖酵母trm 7 Δ突变体：每个突变体都能强烈激活一般氨基酸控制（GAAC） 反应，这是由于不带电荷的tRNA在所有真核生物中的基因表达大规模重新编程， Gcn 2激酶，但trm 7 Δ突变体没有表现出可检测的tRNA充电缺陷。 为了跟进，我们将：1）检查RTD途径和身体改造的相似性和差异 生物学在S. pombe 2）定义在S. 3）定义trm 7 Δ突变体如何激活GAAC途径以及Trm 7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.