Poster abstracts

Poster number 18 submitted by Nripesh Dhungel

tRNA splicing subcellular location: critical or not?

Nripesh Dhungel (The Ohio State University)

Abstract:
Pre-tRNA splicing is critical to the viability of eukaryotic cells. In vertebrates and yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNA is composed of four proteins; Sen2p, Sen15p, Sen34p, and Sen54p. Splicing of pre-tRNA was demonstrated to occur in the nucleus of vertebrates such as Xenopus (Melton, et al., 1980) and humans (Paushkin et al., 2004). In contrast to vertebrate pre-tRNA splicing, yeast pre-tRNA splicing takes place in the cytoplasm on the surface of the mitochondria (Yoshihisa et al., 2003; 2007). The different location of pre-tRNA splicing machinery in yeast versus vertebrate cells raises the question of why the cell biology of this critical process differs between metazoans and yeast. To elucidate the spatial roles of the splicing endonuclease complex, we are directing the localization of the yeast pre-tRNA splicing machinery to the nucleus. To accomplish this, each protein of the splicing machinery has been fused in-frame to a nuclear localization signal and two tandem GFP molecules. To date each of the four components of the splicing endonuclease has been successfully relocated to the nucleus in cells that still contain the endogenous mitochondrial localized complex. Using heterokaryons we showed that none of the four subunits of the endonuclease shuttles out of the nucleus. Similar experiments with the tRNA ligase failed to deliver the majority of the protein into the nucleus. However, we have shown that the protein is not excluded from the nucleus and can still be used for the study. Our next goal is to determine whether the modified proteins maintain catalytic activity. Ultimately, we will determine the physiological consequences of yeast with defective cytosolic splicing machinery and a functional complex in the nucleus. We will accomplish this using either deletions and/or conditional mutations of the genes encoding the endogenous subunits of the splicing endonuclease complex.

Keywords: tRNA splicing