Determining how 2'-O-methylations in the eukaryotic anticodon loop region of tRNA are formed and how they affect translation

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ABSTRACT Translation is a key step in the overall process that cells use to convert genes into proteins, and defects in translation are linked to many human diseases. A critical component of translation is transfer RNA (tRNA), which must be extensively chemically modified by numerous cellular enzymes to function properly. Many of these enzymes and the tRNA modifications that they form are conserved in eukaryotic organisms ranging from single-celled budding yeast to multi-cellular organisms such as humans, thus making yeast a powerful model organism for studying the roles of tRNA modifications. Importantly, defects in tRNA modifications cause diverse neurological disorders such as intellectual disability (ID), and are linked to many other diseases. This NIH R15 AREA proposal seeks to expose undergraduate students to modern biomedical research by advancing the field of tRNA modification research in three ways. First, we propose to study yeast Trm732, a protein which binds to the methyltransferase Trm7 to form a highly conserved modification at position 32 located in the critical anticodon loop (ACL) region of the tRNA. In addition, we will study yeast Trm734, which also binds to Trm7 to form a conserved modification at tRNA position 34 in the ACL. Defects in these modifications due to mutations in human TRM7 (known as FTSJ1) cause ID. Moreover, human TRM732 (THADA) is linked to type 2 diabetes, obesity, and polycystic ovary syndrome, and human TRM734 (WDR6) also has links to human health. Thus, in Aim 1 of the proposal, students will perform biochemical experiments to determine the roles of Trm732 and Trm734 in tRNA modification by Trm7. In our previous round of funding, students identified Trm732 and Trm734 variant proteins lacking tRNA modification activity, which will be valuable tools to understand how each protein functions in tRNA modification. Second, although the cumulative role of these modifications at 32 and 34 have been studied previously, little is known about the individual role of each modification by itself. Thus, in Aim 2 we propose to determine how each modification individually affects translation in yeast, as well as in cultured human and fruit fly cells. Third, we propose to identify the gene required to form a human tRNA ACL modification not found in yeast, and to study its role in translation. In Aim 3 we will finish our work to identify the enzyme that adds a modification to position 39 of certain human, animal, and plant tRNAs. Students will continue to test candidate genes for the modification activity using approaches that we developed previously to identify other tRNA modification genes. Once the gene is identified, we will use reporter assays to determine the role of this modification in translation. The research proposed herein meets the criteria of the NIH R15 AREA award because it gives undergraduate students the opportunity to fully participate in research that will increase knowledge of the underlying molecular causes of disease. Students will be trained in genetic, molecular biological, and biochemical techniques, helping to prepare a new generation of biomedical researchers.