tRNA editing by deamination: Balancing affinity and specificty

描述(申请人提供)：在解码过程中改变tRNA含义的序列改变是越来越多的转录后改变的一部分，统称为tRNA编辑。编辑可以受到编辑站点的结构上下文的影响，在tRNA的情况下，可以通过转录后修饰来调节。在锥虫中，tRNAs在细胞核中转录，输出到细胞质，然后细胞质tRNAs的一个子集被活跃地输入线粒体。然而，在tRNAs能够在任何细胞室中发挥功能之前，它们面临着许多酶反应，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tRNA末端无关序列的修剪过程，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成熟途径的任何点上，也可能发生在任何含有tRNA的间隔中。尽管在过去的几年里取得了很大的进展，但仍然不清楚编辑和修改途径是如何在分子和细胞水平上整合的。我们已经提出，tRNA编辑和修饰事件可以被一些生物高度耦合和利用，以在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，比如锥虫寄生虫，人们普遍认为大部分基因调控发生在转录后。在这项提议中，我们继续了对布鲁氏锥虫非常独特的tRNA编辑酶的研究，但现在在我们新解决的晶体结构的推动下，我们提出了新的问题，即是什么让这种酶如此独特。我们还关注了一组新发现的甲基转移酶，它们出人意料地针对编辑站点，为我们关于编辑和修改的相互关系以及这种耦合在细胞功能方面可能意味着什么的假设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编辑和修饰领域是前所未有的。作为锥虫(利什曼原虫和锥虫)tRNA成熟的重要步骤，这些类型的编辑和修饰事件也为治疗具有重要医学意义的寄生虫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大我们对tRNA在细胞新陈代谢中扮演核心角色的认识。好了！

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. !