Poster abstracts

Poster number 44 submitted by Ernesto Roldan-Bonet

Understanding Transfer RNA Nuclear/Cytoplasmic Transport and Its Role in Quality Control

Ernesto J Roldan-Bonet (Department of Molecular Genetics; Center for RNA Biology; Cellular, Molecular and Biochemical Program; OSU), Paolo L Sinopoli (Department of Molecular Genetics, Center for RNA Biology, OSU)

Abstract:
Transfer RNAs (tRNAs) are essential adaptor molecules that function in translation1. Long believed to move unidirectionally from the nucleus to the cytoplasm in eukaryotic cells, it is now well established that cytoplasmic tRNAs undergo constitutive retrograde nuclear import and re-export2. This widely conserved nuclear retrograde trafficking pathway has been implicated in many important cellular processes including the addition of select post-transcriptional modifications to tRNAs and the regulation of translation under stress conditions. Our work demonstrates that disruption of tRNA nuclear import leads to the accumulation of defective cytoplasmic tRNAs, such as those lacking the post-transcriptional modification m22G26 or bearing unprocessed 5′ leader sequences, implicating nuclear import in the quality control of tRNAs3. Despite this, the fate of aberrant tRNAs following nuclear re-entry and the molecular machinery responsible for their nuclear import remain largely undefined. To address these gaps, we are employing various genetic approaches in the budding yeast Saccharomyces cerevisiae. The possibility of nuclear turnover of hypomodified tRNAs is being assessed by measuring hypomodified tRNA levels in strains lacking known tRNA nuclear turnover components, while nuclear repair is being investigated using a yeast heterokaryon strategy. To identify import factors, we developed a genome-wide screen leveraging the nuclear retrograde pathway dependent wybutosine (yW) modification of tRNAPhe4. Having already screened over 15% of the genome, we have identified proteins known to be involved in yW biosynthesis, validating our approach, and identified additional candidates whose roles in import versus yW biosynthesis are under investigation. Together, these studies will advance our understanding of tRNA retrograde nuclear trafficking and its involvement tRNA quality control.

References:
1)Phizicky, E. M., & Hopper, A. K. (2023). The life and times of a tRNA. https://doi.org/10.1261/rna
2)Shaheen, H. H., & Hopper, A. K. (2005). Retrograde movement of tRNAs from the cytoplasm to the nucleus in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences, 102(32), 11290–11295. https://doi.org/10.1073/pnas.0503836102
3)Kramer, E. B., & Hopper, A. K. (2013). Retrograde transfer RNA nuclear import provides a new level of tRNA quality control in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences, 110(52), 21042–21047. https://doi.org/10.1073/pnas.1316579110
4)Nostramo, R. T., & Hopper, A. K. (2020). A novel assay provides insight into tRNAPhe retrograde nuclear import and re-export in S. cerevisiae. Nucleic Acids Research, 48(20), 11577–11588. https://doi.org/10.1093/nar/gkaa879

Keywords: wybutosine, tRNA retrograde nuclear trafficking, post-transcriptional modifications