Poster abstracts

Poster number 32 submitted by Matt McPherson

Determining 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, which allows cytoplasmic tRNAs to return to the nucleus and is important for modifications and quality control of tRNAs. However, it is currently unknown if tRNAs can undergo retrograde nuclear import more than once. By modifying the synthesis of the wybutosine (yW) modification, which is exclusive to tRNAPhe, we will determine whether the tRNA retrograde pathway is an iterative process. yW is dependent on one full round of nuclear import and re-export, therefore its presence, which the Hopper lab has developed an assay to detect, is indicative of a tRNA undergoing this trafficking process. By making yW synthesis dependent on two rounds of retrograde nuclear import and re-export, tRNAPhe will only receive the modification if this trafficking process is iterative. This can be accomplished by localizing either Tyw2 or Tyw3 into the nucleus, which normally only modify tRNAPhe after re-export into the cytoplasm. We have replaced the endogenous TYW2 and TYW3 genes with constructs that contain the respective ORFs with a 5’ histone nuclear localization signal for nuclear import of the proteins and 3’ GFP for live cell imaging via microscopy. The replacements were accomplished through homologous recombination and confirmed by employing PCR analyses of genomic DNA. Expression of the modified Tyw2/3 GFP-tagged proteins was verified by western blot analysis. For the strain harboring the NLS-TWY2-GFP replacement, Twy2 catalytic activity was confirmed by the presence of yW on endogenous tRNAs. Contingent on the modified Tyw2 protein localizing exclusively to the nucleus, the data support the hypothesis that tRNA retrograde nuclear import is an iterative process; microscopy is currently underway to determine the localization of the proteins

Keywords: tRNA, Nuclear Trafficking, Yeast