Interdisciplinary Graduate Programs Symposium
2012 OSU Molecular Life Sciences
Interdisciplinary Graduate Programs Symposium
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Poster abstracts
Abstract:
tRNA serves an essential function in protein synthesis. Movement of tRNA was thought to be unidirectional from the nucleus to the cytoplasm. However, it is now known that tRNAs move from the cytoplasm to the nucleus via retrograde nuclear import and can again access the cytoplasm via the re-export step. The tRNA retrograde process is conserved from yeast to mammalian cells and is responsive to the nutrient availability. The goal of my research is to elucidate the mechanism of bidirectional tRNA nuclear-cytoplasmic trafficking using yeast as a model system. Our in vivo data show that Msn5, a member of the beta-importin family, is dedicated in the tRNA nuclear re-export step; that is, it exports only spliced tRNA, and perhaps only aminoacylated tRNA (aa-tRNA). However, in vitro studies indicate Msn5 does not have specificity for mature tRNA as Msn5 is able to bind to short double-stranded RNAs. To account for the difference in the in vivo versus in vitro data, I hypothesized that other proteins aid Msn5’s in vivo specificity to recognize and export imported mature tRNA. Previous studies implicated the translation elongation factor Tef1/2 (vertebrate eEF1A) in tRNA export and I showed that Tef1/2 shuttles between the nucleus and the cytoplasm, a prerequisite if Tef1/2 directly functions in the re-export step. Msn5 and Tef1/2 could form an export competent cooperative complex in the nucleus with aa-tRNAs and RanGTP or Tef1/2 might hand off aa-tRNA to Msn5 for re-export without being part of a quaternary shuttling complex. To test these models, I am employing in vivo pull down methodologies and in vitro binding assays. The pull down experiments are conducted using cells with endogenously expressed tagged functional proteins. To maintain the export complex, the cells also have plasmids with inducible Ran locked in the GTP- or the GDP-bound states. My data indicate that Tef1-Myc and Ran co-purify with Msn5 in a Ran-GTP dependent manner supporting the hypothesis that Tef1/2 might assist Msn5 in the tRNA re-export step. Co-purified RNAs with Msn5 are monitored using Northern analyses and RT-PCR. In control experiments, Los1, the tRNA exportin, interacts with both intron-containing and mature tRNAs in a Ran-GTP dependent manner, as anticipated. I am assessing whether Msn5 interacts solely with mature tRNA and/or aa-tRNA.
Keywords: tRNA re-export
