Poster abstracts

Poster number 95 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:
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. This surprising movement event allows tRNAs that have been exported into the cytoplasm to return to the nucleus, after which they can be re-exported back to the cytoplasm to function in protein synthesis. tRNA retrograde nuclear import is necessary for quality control and is required for synthesis of the wybutosine (yW) modification that is exclusive to tRNAPhe. However, it is currently unknown if a tRNA can undergo this trafficking process more than once. By modifying yW synthesis pathway, we can determine whether tRNA retrograde nuclear import and re-export is iterative. Since yW is dependent on one full round of nuclear import and re-export, its presence 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. To accomplish this, we will re-localize either Tyw2 or Tyw3 from the cytoplasm to the nucleus. These proteins normally modify tRNAPhe after it has been re-exported into the cytoplasm, but if they are only localized in the nucleus, a tRNA will only gain yW if it has undergone two rounds of nuclear import and re-export. To visualize potential effects from these modifications, predictions of these modified proteins were created with AlphaFold. These models show that the NLS and GFP do not appear to affect protein folding, with the optimal configuration being with the NLS at the N-terminus and GFP at the C-terminus. Based on this information, we have created TYW2/3 constructs with these specifications. These genes will replace the endogenous loci of TYW2/3 so only the modified genes are present and are expressed at endogenous levels.

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

Keywords: tRNA, Nuclear Trafficking, Yeast