Poster abstracts
Poster number 36 submitted by Matt McPherson
Studies to determine if tRNA retrograde nuclear import is an iterative process
Matthew T. McPherson (The Ohio State University Department of Molecular Genetics), Gabrielle Proctor (University of Southern Utah), Antia K. Hopper (The Ohio State University Department of Molecular Genetics)
Abstract:
For tRNAs to undergo the required post-transcriptional processing steps, they must be transported between the nucleus and cytoplasm. One of these nuclear/cytoplasmic trafficking steps is tRNA retrograde nuclear import, which allows cytoplasmic tRNAs to return to the nucleus and is important for modifications and quality control. However, it is currently unknown if a tRNA can undergo this trafficking process more than once. By modifying the synthesis of the wybutosine (yW) modification, we can determine whether the tRNA retrograde pathway is an iterative process. Since yW is dependent on one full round of nuclear import and re-export, its presence, which can be detected with an assay developed in the Hopper lab, is indicative of a tRNA undergoing this trafficking process. Therefore, by making yW synthesis dependent on two rounds of retrograde nuclear import and re-export, tRNAs will only receive the modification if they can undergo import and re-export more than once. This can be accomplished by localizing either Tyw2 or Tyw3 into the nucleus, which normally only modify tRNAPhe after it has been re-exported. Both Tyw2 and Tyw3 have been successfully localized into the nucleus in independent strains by using the HTB2 nuclear localization signal and GFP for visualization. While our modified proteins have not had their catalytic activity tested, Tyw proteins 1,2, and 4 tagged with GFP are still catalytically active. The modified Tyw2 and Tyw3 are currently overexpressed using an inducible GAL promoter vector so that we will be able to test the stringency of their localization with a heterokaryon assay. Our next steps will be to integrate Tyw2 and Tyw3 at their respective loci for endogenous expression, verify if the modified proteins are catalytically active, and determine if tRNA retrograde nuclear import is iterative an iterative process.
References:
Phizicky EM, Hopper AK. tRNA biology charges to the front. Genes Dev. 24, (17):1832-1860. (2010)
Nostramo, R. T. & Hopper, A. K. A novel assay provides insight into tRNAPhe retrograde nuclear import and re-export in S. cerevisiae. Nucleic Acids Res. 48, 11577–11588 (2020).
Keywords: tRNA, Nuclear Trafficking, Yeast