Expanding the genetic code by targeted pseudouridylation

通过靶向假尿苷化扩展遗传密码

• 批准号: 8419298
• 负责人: YI-TAO YU
• 金额: $ 29.17万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-24 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8419298
• 关键词: Address; Affect; Amino Acids; Anticodon; Attention; Binding; Biological Assay; Biological Process; Boxing; Cells; Clinical; Code; Codon Nucleotides; Collaborations; Complex; Coupled; Data Analyses; Embryo; Enzymes; Family; Future; Genes; Genetic Code; Guide RNA; Hereditary Disease; Human Genetics; In Vitro; Injection of therapeutic agent; Mass Spectrum Analysis; Mediating; Messenger RNA; Microinjections; Modification; Mutation; Nonsense Codon; Nucleotides; Positioning Attribute; Primer Extension; Production; Proteins; Pseudouridine; RNA; RNA, Transfer, Amino Acid-Specific; Reporter; Reporter Genes; Research; Ribosomal RNA; Ribosomes; Role; Sense Codon; Series; Serine-Specific tRNA; Site; Specificity; Staging; Structure; System; Technology; Time; Transfer RNA; Translating; Translations; Uncertainty; Uridine; Wheat; Work; Xenopus oocyte; Yeasts; base; chemical property; clinically relevant; improved; in vivo; member; novel; overexpression; premature; protein purification; research study; success

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. PUBLIC HEALTH RELEVANCE: In this application, we propose to investigate how pseudouridylation of codons affects coding during translation and how widespread naturally occurring pseudouridine is in mRNAs. There is no doubt that completion of the work will greatly enhance our understanding of the roles of RNA modifications in codon recognition, and will dramatically expand the current genetic code. From the clinical perspective, a large number of human genetic diseases are associated with codon mutations, especially those that create a premature nonsense codon. Our preliminary results indicate that targeted pseudouridylation at premature nonsense codons specifically suppresses premature translation termination, thus restoring the production of functional protein. Our application is thus of clinical relevance.

描述（由申请人提供）：拟议的研究重点是 mRNA 假尿苷化及其对编码的影响。假尿苷 (¿) 是修饰最丰富的核苷酸，与所有其他已知的核苷酸不同。我们最近的工作表明，当引入无义密码子和有义密码子时，会导致编码特异性发生变化。我们的初步结果还表明 mRNA 含有天然存在的 ¿我们现在处于独特的位置，可以识别由假尿苷化密码子编码的氨基酸和 tRNA，从而极大地扩展了当前的遗传密码。提出了三个具体目标。目标 1：确定特定假尿苷化有义密码子编码的特定氨基酸在我们最初鉴定 ¿ 介导的编码特异性变化的鼓励下，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苷化有义密码子的氨基酸代码。我们将致力于提高酵母中的假尿苷化效率，从而提高从假尿苷化 mRNA 翻译的蛋白质的产量并促进 MS 测序。如有必要，我们将在非洲爪蟾卵母细胞注射和小麦胚胎体外翻译系统中进行实验，其中假尿苷化的mRNA可以直接用于翻译。我们相信，通过使用这些系统，将会出现完整的氨基酸密码。目标 2：识别识别假尿苷酸化密码子的特定 tRNA 种类（反密码子）。 尽管MS测序方法将揭示氨基酸水平的遗传密码，但从氨基酸密码推导出来的tRNA同工受体家族的哪些成员负责假尿苷化密码子的解码这一问题仍然没有答案。我们建议使用两种独立的方法，即 tRNA 过表达和 tRNA-核糖体-密码子过滤结合，在 tRNA 水平上解码每个假尿苷化密码子。这样做，我们将能够完全破译假尿苷化密码子的遗传密码。特定 tRNA 种类的鉴定将进一步允许合作结晶包含假尿苷化密码子及其新鉴定的同源 tRNA 的核糖体。这样的分析将提供对如何识别假尿苷化密码子的更完整的理解。目标 3：识别 mRNA 中天然存在的基因和细胞中天然的替代编码。 我们的初步实验强烈表明，假尿苷化在 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 中的 mRNA 进行公正的全球搜索了。具体来说，我们将结合 CMC-修饰-引物-延伸和 CLIP 与深度测序，以全局筛选所有 mRNA 中天然存在的 ¿ 内容。然后，将通过最近开发的位点特异性假尿苷化测定来验证和量化由此鉴定的结果。自然发生的识别反过来将使我们能够识别自然发生的替代编码。 公共健康相关性：在本申请中，我们建议研究密码子的假尿苷化如何影响翻译过程中的编码，以及天然存在的假尿苷在 mRNA 中的广泛程度。毫无疑问，这项工作的完成将极大地增强我们对RNA修饰在密码子识别中的作用的理解，并将极大地扩展当前的遗传密码。从临床角度来看，大量人类遗传疾病与密码子突变有关，尤其是那些产生过早无义密码子的突变。我们的初步结果表明，过早无义密码子的靶向假尿苷化特异性抑制过早翻译终止，从而恢复功能蛋白的产生。因此，我们的应用具有临床相关性。