Poster abstracts
Poster number 20 submitted by Jackson Hastings
Studies to Identify tRNA Nuclear Export Adaptors in Saccharomyces cerevisiae
Jackson E. Hastings (Department of Molecular Genetics, The Ohio State University), Sophie Cihan (Department of Molecular Genetics, The Ohio State University), Anita K. Hopper (Department of Molecular Genetics, The Ohio State University)
Abstract:
Transfer RNAs (tRNAs) deliver amino acids to the ribosome and are essential for protein synthesis in all living organisms. Three karyopherins share the function of primary tRNA nuclear export in budding yeast: Los1, Mex67-Mtr2, and Crm1. Los1 directly binds tRNA and Ran-GTP to form a tRNA nuclear export complex. However, the mechanisms of complex assembly for Crm1 and Mex67-Mtr2 remain unknown. Crm1 and Mex67-Mtr2 generally employ proteins that function as adaptors, allowing for indirect binding of their various RNA cargoes. We hypothesize that unidentified Saccharomyces cerevisiae proteins function as adaptors for Mex67-Mtr2 and Crm1 by binding both intron-containing pre-tRNA cargoes and their respective exporter. Employing streptavidin magnetic particles conjugated to biotinylated oligonucleotides with sequence complementarity to specific intron-containing tRNAs, we can study export complexes at the primary tRNA nuclear export step. We used an RNA pulldown technique to isolate individual tRNA species from a pool of all tRNAs, enriching for 7 of the 10 budding yeast intron-containing tRNAs. We also isolated pre-tRNAIleUAU from cell extracts in which nuclear export complexes were enriched using an inducible Ran mutant locked in its active GTP form. Using this strategy, we copurified Crm1-GFP from tRNA pulldown samples, as confirmed by western blot analysis; enriched nuclear export complexes were then subjected to mass spectrometry (MS). Since export adaptors must bind exporter and tRNA simultaneously, we compared the MS hits to the list of known exporter binding proteins to create a shortlist of adaptor candidates. To confirm the functions of these candidates in primary tRNA nuclear export, we will conduct FISH analyses in wildtype versus mutant adaptor candidate cells. Identification of all tRNA nuclear export complex components is integral to the understandings of their functions and the potential regulatory pathways that govern cytoplasmic tRNA abundance.
References:
1. Chatterjee K, Marshall WA, Hopper AK. Three tRNA nuclear exporters in S. cerevisiae: parallel pathways, preferences, and precision. Nucleic Acids Res. 2022 Sep 23;50(17):10140-10152. doi: 10.1093/nar/gkac754. PMID: 36099418; PMCID: PMC9508810.
2. Engel SR, Aleksander S, Nash RS, Wong ED, Weng S, Miyasato SR, Sherlock G, Cherry JM. Saccharomyces Genome Database: Advances in Genome Annotation, Expanded Biochemical Pathways, and Other Key Enhancements. Genetics. 2024 Nov 12:iyae185. doi: 10.1093/genetics/iyae185. Epub ahead of print. PMID: 39530598.
Keywords: tRNA, nucleus, transport