Poster abstracts

Poster number 31 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), Anita K. Hopper (The Ohio State University Department of Molecular Genetics)

Abstract:
Transfer RNAs (tRNAs) are small non-coding RNAs that are essential for protein synthesis. tRNAs are transcribed and processed in the nucleus before being exported into the cytoplasm for further processing, including splicing (in non-vertebrate organisms). Cytoplasmic tRNAs return to the nucleus via tRNA retrograde nuclear import. This trafficking process serves multiple functions including proper tRNA modification. Imported tRNAs are re-exported to the cytoplasm where they function in protein synthesis. However, it is currently unknown if a tRNA can undergo nuclear import more than once. If the tRNA retrograde pathway is an iterative process, it could function as a novel regulator of protein synthesis. To answer this question, we are utilizing the tRNAPhe specific modification wybutosine (yW), which is dependent on retrograde nuclear import and re-export. After splicing and retrograde nuclear import, tRNAPhe is modified by Trm5 at position G37. tRNAPhe is re-exported to the cytoplasm and sequentially modified by Tyw1,2,3, and 4 at position G37 resulting in the yW modification. yW can be detected via an assay developed by the Hopper lab. To determine if tRNA nuclear import and re-export is iterative, we devised a methodology to modify the yW synthesis pathway. Since the Tyw proteins sequentially modify tRNAPhe if either Tyw2 or Tyw3 is nuclear, tRNAPhe will only receive yW upon two full rounds of nuclear import and re-export. We have relocated Tyw3 with an N-terminus nuclear localization signal (NLS) and a C-terminus GFP for live cell microscopy. Yeast strains containing the NLS-TYW3-GFP were generated by either replacing the endogenous TYW3 or by expression via a galactose-inducible plasmid-encoded NLS-TYW3-GFP in a tyw3Δ strain. Microscopy and heterokaryon analysis show that NLS-Tyw3-GFP is nuclear. The HCl/aniline assay of both strains resulted in yW modification of tRNAPhe, suggesting that tRNA nuclear import and re-export is an iterative process.

References:
Hopper AK, Nostramo RT. tRNA Processing and Subcellular Trafficking Proteins Multitask in Pathways for Other RNAs Front Genet 2019 10:96.

Nostramo RT, Hopper AK. 2020 A novel assay provides insight into tRNAPhe retrograde nuclear import and re-export in S. cerevisiae. Nucleic Acids Res. 18;48(20):11577-11588

Keywords: tRNA, Nuclear Trafficking, Yeast