Expanding the genetic code by targeted pseudouridylation

通过靶向假尿苷化扩展遗传密码

• 批准号: 8720024
• 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/8720024
• 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; deep sequencing; 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.

描述（由适用提供）：拟议的研究重点介绍mRNA pseudouridylation及其对编码的影响。伪苷（€）是最彻底修饰的核苷酸，与所有其他已知的核苷酸不同。我们最近的工作表明，当被引入胡说八道和感知密码子时，会导致编码特异性变化。我们的初步结果还表明mRNA含有自然存在的„。现在，我们处于一个独特的位置，可以识别由伪基的密码子编码的氨基酸和TRNA，从而大大扩展了当前的遗传密码。提出了三个具体目标。目标1：要确定特定的氨基酸，并通过我们对`介导的编码特异性变化的最初识别而鼓励的特定假尿素的感觉密码子，我们建议在体内和体外翻译中使用质谱法（MS）测序，以定义氨基酸代码，以定义氨基酸代码含量的代码，使所有PseudiudiRidyl senseyls senseyls senseyls senseyls senseyls senseyls senseyls sense sense sensement senseyls senseyls senseyls senseyls sense sense consementys copecasts code code。我们将致力于提高酵母中的假胞苷化效率，从而增强从假基因甲基化mRNA转化并支持MS测序的蛋白质的产生。如有必要，我们将在爪蟾卵母细胞注射和小麦胚胎体外翻译系统中进行实验，其中伪基的mRNA可直接用于翻译。我们认为，通过使用这些系统，将出现完整的氨基酸代码。目的2：确定识别假胞醇化密码子的特定tRNA物种（盘子源）。尽管MS测序方法将在氨基酸水平上揭示遗传密码，但根据氨基酸代码推论的tRNA等ancceptor家族的哪个成员的问题负责解码假纤维化的密码子仍未得到解答。我们建议使用两种独立的方法，即tRNA的过表达和tRNA-核糖体 - 孔子滤光片结合，以在tRNA级别（每个假尿素化密码子）下解码。这样，我们将能够完全破译假胞苷二基化密码子的遗传密码。特定tRNA物种的鉴定将进一步允许协作努力，以使含有假素化密码子及其新识别的同源TRNA的核糖体结晶。这样的分析将对如何识别假性代码子进行更完整的了解。目标3：确定在mRNA中自然存在的s和细胞中天然替代编码。我们的初步实验强烈表明伪啶基化自然发生在mRNA中。我们认为，在现代技术的帮助下 - 深入的测序，是时候在mRNA中进行公正的全球搜索。具体而言，我们将结合CMC-Modification-Primer-Extension和Clip与深层测序，以全球筛选所有mRNA，以自然发生。因此，将通过最近开发的位点特异性假基因化测定法对确定的S进行验证和量化。天然发生�S的识别反过来将使我们能够识别自然发生的替代编码。