tRNA editing by deamination: Balancing affinity and specificty

描述（由申请人提供）：在解码过程中改变tRNA意义的序列改变是越来越多的转录后变化的一部分，统称为tRNA编辑。编辑可以受到编辑位点结构背景的影响，在tRNA的情况下，可以通过转录后修饰进行调节。在锥虫中，trna在细胞核中转录，输出到细胞质，然后细胞质中的一部分trna被主动输入到线粒体中。然而，在tRNA能够在任何细胞区室中发挥功能之前，它们面临许多酶促反应，包括末端修剪、内含子剪接、tRNA编辑和化学修饰。其中一些过程，例如那些涉及修剪tRNA末端的外来序列的过程，发生在细胞核中，通常在细胞质输出之前。其他的，如编辑和修饰，可能发生在tRNA成熟途径的任何点和任何含有tRNA的区室中。尽管过去几年取得了很大进展，但编辑和修饰途径如何在分子和细胞水平上整合仍然不清楚。我们提出tRNA编辑和修饰事件可以高度耦合，并被一些生物体利用，在tRNA特异性水平上控制基因表达。这在单细胞真核生物中尤其重要，如锥虫寄生虫，人们普遍认为大部分遗传调控发生在转录后。在这个提案中，我们继续我们对T. brucei非常独特的tRNA编辑酶的研究，但现在由于我们新解决的晶体结构，我们提出了新的问题，是什么使这种酶如此独特。我们还关注了一组新发现的甲基转移酶，它们令人惊讶地靶向编辑位点，为我们关于编辑和修饰相互关系的假设以及这种耦合在细胞功能方面的意义提供了一个很好的试验场。值得注意的是，体外甲基化活性的重建需要添加重组编辑酶，这在tRNA编辑和修饰领域是没有先例的。作为锥虫病（利什曼原虫和锥虫）中tRNA成熟的重要步骤，这些类型的编辑和修饰事件也为针对具有重要医学意义的寄生虫的治疗干预提供了非常有吸引力的靶点。鉴于tRNA成熟与疾病之间的联系，这些研究将进一步扩展我们对tRNA在细胞代谢中扮演核心角色的认识。！

DESCRIPTION (provided by applicant): Sequence alterations that change the meaning of a tRNA in decoding are part of a growing number of post-transcriptional changes collectively known as tRNA editing. Editing can be influenced by the structural context of an editing site and in the case of tRNA can be modulated by posttranscriptional modifications. In trypanosomatids, tRNAs are transcribed in the nucleus, exported to the cytoplasm, and later a subset of cytoplasmic tRNAs is actively imported into the mitochondria. However, before tRNAs can be rendered functional in any cellular compartment, they face many enzymatic reactions including end trimming, intron splicing, tRNA editing, and chemical modification. Some of these processes, for example those involved in trimming of extraneous sequences at the tRNA ends, occur in the nucleus, usually preceding cytoplasmic export. Others, like editing and modification, may occur at any point in the tRNA maturation pathway and in any of the tRNA-containing compartments. Despite much progress made in last few years, it is still not clear how editing and modification pathways are integrated both at the molecular and cellular levels. We have proposed that tRNA editing and modification events can be highly coupled and exploited by some organisms to control gene expression at the level of tRNA specificity. This is especially important in single-cell eukaryotes like trypanosomatid parasites where is well accepted that the bulk of the genetic regulation occurs post-transcriptionally. In this proposal, we have continued our studies on the very unique tRNA editing enzyme of T. brucei but now prompted by our newly solved crystal structure we ask new questions of what makes this enzyme so unique. We also focus on a newly discovered set of methyltransferases, which surprisingly target the editing site providing a wonderful testing ground for our hypothesis of the interrelation of editing and modification and what this coupling may mean in terms of cellular function. Significantly, reconstitution of methylation activity in vitro requires addition of the recombinant editing enzyme a finding that is without precedent in the tRNA editing and modification field. As essential steps in tRNA maturation in trypanosomatids (Leishmania and Trypanosoma), these types of editing and modification events also provide very attractive targets for therapeutic intervention against parasites of very major medical importance. Given the link between tRNA maturation and disease, these studies will further expand our knowledge of the role tRNA takes as a central player in cellular metabolism. !