Poster abstracts

Poster number 17 submitted by Hsiao-Yun Huang

Regulation of tRNA Bidirectional Nuclear-cytoplasmic Trafficking in Saccharomyces cerevisiae

Hsiao-Yun Huang (MCDB, Department of Molecular Genetics, the Ohio State University), Anita K. Hopper (Department of Molecular Genetics, the Ohio State University)

Abstract:
Movement of tRNA was thought to be unidirectional from nucleus to cytoplasm. However, the tRNA retrograde process was discovered by which tRNAs move from the cytoplasm to the nucleus via retrograde nuclear import and can again access the cytoplasm via the tRNA re-export step. The conserved tRNA retrograde process is responsive to environmental conditions, in particular, nutrient availability. It is likely a newly discovered mechanism by which cells control gene expression in response to nutrients. The aim of this research is to understand the mechanisms of tRNA nuclear-cytoplasmic trafficking. Msn5, a member of the beta-importin family, appears to be dedicated to the tRNA re-export step. Since in vivo data indicate that Msn5 exports only spliced tRNA, and perhaps only aminoacylated tRNA, but in vitro Msn5 binds short duplex (e.g. microRNAs), we hypothesize that other proteins aid Msn5’s in vivo specificity to recognize mature tRNA. Previous studies implicated that the translation factor, Tef1/2 (eEF1A in vertebrates) may function in the tRNA re-export step and we also showed that Tef1/2 shuttles between the nucleus and the cytoplasm supporting the prerequisite for Tef1/2 subcellular dynamics if Tef1/2 were to function in the re-export step. Thus, I am performing biochemical studies to test whether Msn5 and Tef1/2 form an export competent cooperative complex in the nucleus with aminoacylated-tRNAs (aa-tRNAs) and RanGTP, or whether Tef1/2 delivers aa-tRNAs to Msn5 for tRNA re-export. My preliminary data show that Tef1-Myc and Ran co-purify with Msn5 in the cell extracts with an inducible RanGTP-locked form, but not in cells with Ran in the GDP-locked form, suggesting that Msn5 interacts with Tef1 and that the export complex is likely maintained in my experimental conditions. I will determine whether mature or charged tRNA co-purify with Msn5 in the complex by Northern analyses and investigate the specificity of Msn5 in vitro to recognize mature and/or aa-tRNAs.

References:
1. Hopper, A.K. and H.H. Shaheen, A decade of surprises for tRNA nuclear-cytoplasmic dynamics. Trends Cell Biol, 2008. 18(3): p. 98-104.
2. Murthi, A., et al., Regulation of tRNA bidirectional nuclear-cytoplasmic trafficking in Saccharomyces cerevisiae. Mol Biol Cell, 2010. 21(4): p. 639-49

Keywords: tRNA re-export