Poster abstracts
Poster number 58 submitted by Jackson Hastings
Identification of tRNA export adaptors in Saccharomyces cerevisiae
Jackson Hastings (Department of Molecular Genetics, The Ohio State University), Anita K. Hopper (Department of Molecular Genetics, The Ohio State University)
Abstract:
Transfer RNAs (tRNAs) are biomolecules that deliver amino acids to the ribosome and are essential components for protein synthesis in all living things. In eukaryotes, the nuclear export of tRNAs, which is required to enable tRNA access to the translation machinery, is a function that is shared by three nuclear exporters in budding yeast: Los1, Mex67-Mtr2, and Crm1. However, while Los1 binds tRNA directly, Mex67-Mtr2 and Crm1 do not, suggesting that the latter two exporters instead employ the use of yet unknown protein adaptors to indirectly bind tRNA. I hypothesize that certain Saccharomyces cerevisiae proteins act as adaptors for Crm1 and Mex67 by binding both tRNA cargo and their respective exporter. Therefore, I am conducting serial tandem affinity purification assays on yeast lysate – first pulling down tagged exporter proteins and then the tRNAs in complex with them – to isolate proteins that bind both exporter and tRNA simultaneously. Mass spectrometry on the resulting isolate will provide a shortlist of adaptor candidates that will then be verified by Northern blot analysis. I have successfully executed both component parts of the tandem affinity purification individually. Using magnetic beads conjugated to anti-GFP, I have isolated tRNA nuclear export complexes involving Crm1 (tagged with GFP at its endogenous locus), confirming the presence of both Crm1 protein and its tRNA cargo via Western Blot and RT-PCR, respectively. I have also used biotinylated DNA probes (each complimentary to a specific tRNA intron sequence) and streptavidin magnetic beads to isolate specific species of tRNAs from a pool of multiple tRNAs. Current efforts involve executing the two assays in sequence to isolate tRNA nuclear export adaptor candidates.
References:
Wu J, Bao A, Chatterjee K, Wan Y, Hopper AK. Genome-wide screen uncovers novel pathways for tRNA processing and nuclear-cytoplasmic dynamics. Genes Dev. 2015 Dec 15;29(24):2633-44. doi: 10.1101/gad.269803.115. PMID: 26680305; PMCID: PMC4699390.
Chatterjee K, Majumder S, Wan Y, Shah V, Wu J, Huang HY, Hopper AK. Sharing the load: Mex67-Mtr2 cofunctions with Los1 in primary tRNA nuclear export. Genes Dev. 2017 Nov 1;31(21):2186-2198. doi: 10.1101/gad.305904.117. Epub 2017 Dec 6. PMID: 29212662; PMCID: PMC5749166.
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.
Keywords: tRNA, budding yeast, nuclear export