Expanding the genetic code by targeted pseudouridylation

描述(申请人提供)：建议的研究重点是信使核糖核酸的假尿路作用及其对编码的影响。假尿苷(�)是修饰最丰富的核苷酸，不同于所有已知的核苷酸。我们最近的工作表明，当�被引入无义和有义密码子中时，会导致编码特异性的变化。我们的初步结果还表明，基因中含有自然产生的�S。我们现在处于一个独特的位置，可以识别由假尿路密码子编码的氨基酸和tRNA，从而极大地扩展了当前的遗传密码。提出了三个具体目标。目的1：为了确定�介导的编码特异性变化所鼓励的特定假尿苷酸化正义密码子编码的特定氨基酸，我们建议使用体内和体外翻译结合质谱分析(MS)来定义包含所有伪尿苷酸化正义密码子的氨基酸编码。我们将致力于提高酵母中的假尿苷酸化效率，从而提高从假尿苷酸化的mRNAs翻译的蛋白质的产量，并促进MS测序。如有必要，我们将在非洲爪哇卵母细胞注射和小麦胚胎体外翻译系统中进行实验，在该系统中可以直接使用假性尿苷酸化的mRNA进行翻译。我们相信，通过使用这些系统，一个完整的氨基酸编码将会出现。目的2：鉴定识别假尿苷酸化密码子的特定tRNA物种(反密码子)。虽然MS测序方法将揭示氨基酸水平的遗传密码，但从氨基酸密码推断的tRNA等受体家族中的哪个成员(S)负责破译假尿苷酸密码子的问题仍未得到回答。我们建议使用两种独立的方法，即tRNA过度表达和tRNA-核糖体-密码子过滤器结合，在tRNA水平上解码每个假尿苷酸化密码子。通过这样做，我们将能够完全破译假尿路密码子的遗传密码。对特定tRNA物种的鉴定将进一步允许合作努力使包含假尿路密码子及其新发现的同源tRNA的核糖体结晶。这样的分析将提供一个更完整的理解如何识别伪偶联密码子。目的3：鉴定mRNAs中自然存在的�S及其在细胞中的自然替代编码。我们的初步实验有力地表明，假尿路连接在mRNAs中自然发生。我们认为，在现代技术--深度测序的帮助下，现在是时候在mRNAs中进行一次公正的全球搜索�S了。具体地说，我们将结合CMC-修饰-引物-延伸和CLIP与深度测序来全局筛选所有自然产生的�内容的mRNA。由此确定的�S随后将通过最近发展的位点特异性假尿嘧啶核苷分析进行验证和定量。识别自然产生的�S将反过来使我们能够识别自然产生的替代编码。

DESCRIPTION (provided by applicant): The proposed research focuses on mRNA pseudouridylation and its effect on coding. Pseudouridine (�) is the most abundantly modified nucleotide, which is distinct from all other known nucleotides. Our recent work indicates that �, when introduced into nonsense and sense codons, results in coding specificity changes. Our preliminary results also suggest that mRNA contains naturally-occurring �s. We are now in a unique position to identify amino acids and tRNAs that are coded for by pseudouridylated codons, thereby dramatically expanding the current genetic code. Three specific aims are proposed. Aim 1: To determine specific amino acids encoded by specific pseudouridylated sense codons Encouraged by our initial identification of �-mediated coding specificity changes, we propose to use in vivo and in vitro translation coupled with mass spectrometry (MS) sequencing to define an amino acid code encompassing all pseudouridylated sense codons. We will work towards improving pseudouridylation efficiency in yeast, thus enhancing the production of proteins translated from pseudouridylated mRNAs and facilitating MS sequencing. If necessary, we will carry out the experiments in the Xenopus oocyte injection and the wheat embryo in vitro translation systems, in which pseudouridylated mRNA can be directly used for translation. We believe that through the use of these systems, a complete amino acid code will emerge. Aim 2: To identify specific tRNA species (anticodons) that recognize pseudouridylated codons. Although the MS sequencing approach will reveal the genetic code at the amino acid level, the question of which member(s) of the tRNA isoacceptor families, deduced from the amino acid code, is responsible for the decoding of a pseudouridylated codon remains unanswered. We propose to use two independent approaches, namely, tRNA over-expression and tRNA-ribosome-codon filter-binding, to decode, at the tRNA level, each pseudouridylated codon. In doing so, we will be able to fully decipher the genetic code of pseudouridylated codons. The identification of specific tRNA species will further allow a collaborative effort to crystallize the ribosomes that contain pseudouridylated codons and their newly-identified cognate tRNAs. Such analyses will provide a more complete understanding of how pseudouridylated codons are recognized. Aim 3: To identify naturally occurring �s in mRNAs and natural alternative coding in cells. Our preliminary experiments strongly suggest that pseudouridylation occurs naturally in mRNAs. We believe that, with the help of modern technology--deep sequencing, it is time to conduct an unbiased global search for �s in mRNAs. Specifically, we will combine CMC-modification-primer-extension and CLIP with deep sequencing to globally screen all mRNAs for naturally occurring � content. �s thus identified will then be verified and quantified by recently developed site-specific pseudouridylation assays. The identification of naturally occurring �s will in turn allow us to identify naturally occurring alternative coding.