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.
