Poster abstracts
Poster number 180 submitted by Ciara Balanza
Identifying nuclear localization signals of the human tRNA Splicing Endonuclease Complex (TSEN)
Ciara Balanza (Department of Biochemistry and Molecular Biology, University of Chicago), Cassandra Hayne (Department of Biochemistry and Molecular Biology, University of Chicago)
Abstract:
The splicing of introns from intron containing tRNAs is an essential cellular process. Despite the importance of this process, the human tRNA splicing endonuclease (TSEN) has conflicting evidence regarding subcellular localization. One study reports nuclear localization, while a pre-print suggests presence in the cytoplasm. Understanding the localization of TSEN is integral to understanding the overall regulation of TSEN and tRNA splicing, since mutations in TSEN are linked to Pontocerebellar Hypoplasia (PCH), a rare neurodevelopmental disease. Since reliable antibodies remain a major barrier for clarifying this mystery, we took a subunit focused approach. We predicted nuclear localization signals (NLS) in three TSEN subunits, supporting the idea that TSEN could be nuclear. We hypothesize that one or more of these NLS’s could be necessary or sufficient for driving TSEN to the nucleus.
To test if the NLS’s we identified were sufficient for nuclear localization, we engineered GFP constructs with predicted short NLS signals from TSEN2, TSEN34, and a large disordered region of TSEN54, which contains the predicted NLS. These plasmids were transfected into cells to observe if they were sufficient to drive nuclear localization, compared with a GFP control plasmid. Microscopy results showed the NLS-containing region of TSEN54 had the strongest nuclear localization out of the partial TSEN subunits. To test if this region of TSEN54 is necessary for nuclear localization, we generated a TSEN54-GFP construct missing this region. Our data suggests the deletion of this region results in a loss of localization to the nucleus. Thus, our experiments suggest that the strongest NLS is within the disordered region of TSEN54. Intriguingly, literature indicates that the disordered region of TSEN54 is also the binding site of CLP1, a kinase which associates with TSEN. Therefore, a future direction of our work is to examine the interplay between CLP1 and TSEN in nuclear localization.
Keywords: