Investigating human transfer RNA variants that cause errors in protein synthesis

所有细胞都含有一组转移RNA(TRNAs)，它们是连接基因中的核酸序列和蛋白质中的氨基酸序列的分子适配器。氨酰-tRNA合成酶负责将特定的tRNA与正确的氨基酸连接起来。在所有活的细胞中，蛋白质编码基因被转录成信使RNA(Messenger RNAs，mRNAs)，其中的三核苷酸序列称为密码子，由适当的tRNA的反密码子通过核糖体上的碱基配对来合成确定序列的蛋白质。与加州大学圣克鲁斯分校(UCSC)的Todd Lowe博士的新国际合作将与我的实验室在tRNA生物学方面的专业知识产生协同效应，以确定自然发生的tRNA变体如何影响人类细胞中的蛋白质合成。洛博士通过将人类基因变异与单个tRNA基因相关联，揭示了我们人群中tRNA突变的多样性，震惊了整个研究界。该项目将(目标1)表征自然产生的tRNA变体通过在一组密码子上加入错误的氨基酸而导致功能障碍的能力，并(目标2)确定tRNA基因上游和下游区域如何影响表达水平。由于人类编码600多个tRNA基因，我们的研究将首次使用tRNA变体将其他相同的tRNA转录本映射到特定的tRNA基因。我的实验室将确定人类tRNA变体对蛋白质合成的影响，而洛博士的实验室将贡献最先进的tRNA测序，以精确量化细胞中突变tRNA的水平。预期的结果将有助于通过揭示人类细胞出人意料的能力来容忍蛋白质合成中的错误，从而促进重大科学突破。TRNA水平的定量对于确定许多人类tRNA基因的功能是必不可少的，并且对研究目标至关重要。在加拿大，目前还不存在获得tRNA测序专业知识的机会。这项工作将把新的尖端生物技术引入加拿大，并在正常细胞中扩大关于人类tRNA生物学的基础知识，从而使加拿大人受益。

All cells contain a set of transfer RNAs (tRNAs), which are molecular adaptors connecting the sequences of nucleic acids in genes to the sequences of amino acids in proteins. The aminoacyl-tRNA synthetases are responsible for ligating a specific tRNA with the correct amino acid. In all living cells, protein coding genes are transcribed into messenger RNAs (mRNAs), in which tri-nucleotide sequences, called codons, are decoded by the anticodon of the appropriate tRNA via base pairing on the ribosome to synthesize proteins of defined sequence. The new international collaboration with Dr. Todd Lowe at the University of California Santa Cruz (UCSC) will generate synergy with my lab's expertise in tRNA biology to determine how naturally occurring tRNA variants effect protein synthesis in human cells. Dr. Lowe stunned the research community by mapping human genetic variants to individual tRNA genes, revealing a diversity of tRNA mutations in our population. This project will (aim 1) characterize the ability of naturally occurring tRNA variants to cause malfunction by incorporating the wrong amino acid at a set of codons and (aim 2) determine how the regions up and down stream of tRNA genes impact expression levels. Because humans encode over 600 tRNA genes, our studies will use tRNA variants to map otherwise identical tRNA transcripts to specific tRNA genes for the first time. My lab will determine the impact of human tRNA variants on protein synthesis, while Dr. Lowe's lab will contribute state-of-the-art tRNA sequencing to precisely quantify the levels of mutant tRNAs in cells. The anticipated outcomes will contribute to a major scientific breakthrough by revealing the unexpected ability of human cells to tolerate errors in protein synthesis. Quantitation of tRNA levels is essential to determine the function of the many human tRNA genes and critical to the research objectives. Access to tRNA sequencing expertise is currently non-existent in Canada. The work will benefit Canadians by importing new and cutting-edge biotechnology to Canada and expanding fundamental knowledge about human tRNA biology in normal cells.