tRNA editing by deamination: Balancing affinity and specificity

遗传密码的简并性意味着需要61个意义密码子来指定20种不同的氨基酸，并且除了蛋氨酸和色氨酸外，每种氨基酸都由多个密码子编码。密码子和氨基酸数量之间的差异首先由克里克的摆动假说解释，该假说在解码过程中调用了第一个反密码子和第三个密码子位置之间的灵活性。自摆动规则开始以来，已经描述了100多种转录后修饰，其中影响tRNA反密码子的修饰最多。随着反密码子修饰的积累，新的发现导致了对摆动规则的不断重新解释，包括对tRNA功能的新影响。扩大解码能力的反密码子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。我们认为，tRNA编辑提供了一种有效调节遗传密码退化的机制。编辑还可以用于调节基因表达。此外，编辑本身可以受到编辑位点的结构背景的影响，并且在tRNA的情况下可以通过转录后修饰进行调节。在本文中，我们主要研究了锥虫trna中肌苷的形成过程。我们在单个tRNA中发现了两个编辑事件的第一个例子，其中tRNAThr的位置32和34分别经历了C到U和a到I的编辑。每个含有tRNA的肌苷在32位（摆动位置的5'）也经历了C到U的编辑，这一发现提出了两个编辑事件在该tRNA的功能中起什么作用的重要问题。通过建立体外A to I编辑实验，我们已经证明C to U可以刺激体外A to I编辑。我们还确定了负责A到I编辑的酶，并表明该酶的一个独特特征是它能够进行两种不同的脱氨反应，并且能够利用DNA和RNA作为底物。因此，该提案将专注于回答指定A到I编辑的机制性质的具体问题，并定义这些酶如何实现其特异性。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加工在细胞功能中所起作用的认识。公共卫生相关性：利什曼原虫属和锥虫属感染了全世界数百万人。在这些生物体中，trna经历了该系统特有的转录后编辑变化。在锥虫中负责tRNA编辑变化的酶具有与该蛋白家族的任何其他成员不共享的底物特异性。因此，确定这些酶的哪些特征赋予它们独特的特异性是很重要的。所提出的研究将确定布鲁氏杆菌编辑酶的底物区分的基础，这可能在未来为开发针对这一重要活性的药物打开大门。这些研究也将为胞苷脱氨酶（CDA）超家族的重要成员提供功能和进化的见解。

DESCRIPTION (provided by applicant): Summary The degeneracy of the genetic code is implied in the need for 61 sense codons to specify 20 different amino acids and, with the exception of methionine and tryptophan, each amino acid is encoded by more than one codon. This discrepancy between codon and amino acid numbers was first explained by Crick's wobble hypothesis, which invoked flexibility between the first anticodon and third codon positions during decoding. Since the inception of the wobble rules, over 100 posttranscriptional modifications have been described with the largest number affecting the anticodon of tRNA. As anticodon modifications accrue, new findings lead to a constant reinterpretation of the wobble rules to include novel effects on tRNA function. Anticodon-sequence alterations that expand decoding capacity are part of a growing number of post-transcriptional changes collectively known as tRNA editing. It is our view that tRNA editing provides a mechanism to effectively accommodate genetic code degeneracy. Editing can also be utilized to regulate gene expression. Furthermore, editing itself can be influenced by the structural context of an editing site and in the case of tRNA can be modulated by posttranscriptional modifications. In this proposal, we have focused on the process of inosine formation in the tRNAs of trypanosomatids. We have discovered the first example of two editing events in a single tRNA, whereby positions 32 and 34 of tRNAThr undergo C to U and A to I editing respectively. The finding that every inosine containing tRNA also undergoes C to U editing at position 32 (5' of the wobble position) raises important questions as to what role the two editing events play in the function of this tRNA. By establishing an in vitro A to I editing assay, we have demonstrated that C to U stimulates A to I editing in vitro. We have also identified the enzyme responsible for A to I editing and shown that a unique feature of this enzyme is its ability to perform two different deamination reactions and also is able to utilize both DNA and RNA as substrates. This proposal will thus focus on answering the specific questions of the nature of the machinery that specifies A to I editing and define how these enzymes achieve their specificity. As an essential step in tRNA maturation in trypanosomatids (Leishmania and Trypanosoma), these types of editing also provide a very attractive target 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 processing plays in cellular function. PUBLIC HEALTH RELEVANCE: Members of the genus Leishmania and Trypanosoma infect millions of people worldwide. In these organisms, tRNAs undergo post-transcriptional editing changes that are unique to this system. The enzyme responsible for tRNA editing changes in trypanosomatids possesses substrate specificities that are not shared with any other member of this family of proteins. It is thus important to define what features of these enzymes give them their unique specificity. The propose studies will determine the basis for substrate discrimination of the T. brucei editing enzyme which in the future may open doors towards the development of drugs against this essential activity. These studies will also provide functional and evolutionary insights into important members of the cytidine deaminase (CDA) superfamily.