Expanding the genetic code by targeted pseudouridylation

描述（由申请人提供）：拟研究的重点是mRNA假尿嘧啶化及其对编码的影响。伪尿嘧啶是最丰富的核苷酸修饰，这是不同于所有其他已知的核苷酸。我们最近的工作表明，当将¿引入无义密码子和有义密码子时，会导致编码特异性的变化。我们的初步结果还表明mRNA含有自然产生的¿s。我们现在处于一个独特的位置，可以识别由假尿苷化密码子编码的氨基酸和trna，从而极大地扩展当前的遗传密码。提出了三个具体目标。目的1：为了确定由特定假尿嘧啶化意义密码子编码的特定氨基酸，我们最初确定了介导的编码特异性变化，我们建议使用体内和体外翻译结合质谱（MS）测序来定义包含所有假尿嘧啶化意义密码子的氨基酸密码子。我们将致力于提高酵母的假尿嘧啶化效率，从而提高假尿嘧啶化mrna翻译蛋白的产量，并促进质谱测序。如有必要，我们将在爪蟾卵母细胞注射和小麦胚体外翻译系统中进行实验，可以直接使用假尿嘧啶化mRNA进行翻译。我们相信，通过使用这些系统，一个完整的氨基酸代码将出现。目的2：鉴定识别假尿嘧啶化密码子的特定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出的tRNA同受体家族的哪个成员负责解码假尿嘧啶化密码子的问题仍然没有答案。我们建议使用两种独立的方法，即tRNA过表达和tRNA-核糖体-密码子过滤器结合，在tRNA水平上解码每个假尿嘧啶化密码子。这样，我们将能够完全破译假尿嘧啶化密码子的遗传密码。特定tRNA物种的鉴定将进一步允许合作努力结晶含有假尿嘧啶化密码子的核糖体及其新鉴定的同源tRNA。这样的分析将提供一个更完整的理解如何识别假尿嘧啶化密码子。目的3：鉴定mrna中自然发生的编码和细胞中自然的替代编码。我们的初步实验强烈表明，假尿嘧啶化在mrna中自然发生。我们相信，在现代技术——深度测序的帮助下，是时候对mrna进行公正的全球搜索了。具体而言，我们将结合cmc -修饰-引物-延伸和CLIP与深度测序，以全球筛选所有天然存在的mrna。然后，通过最近开发的位点特异性伪尿嘧啶化测定法对鉴定出的基因进行验证和量化。对自然发生的编码的识别反过来又使我们能够识别自然发生的替代编码。

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.