Poster abstracts
Poster number 33 submitted by Matt McPherson
Is tRNA retrograde nuclear import and re-export an iterative process?
Matthew T. McPherson (The Ohio State University Department of Molecular Genetics), Anita K. Hopper (The Ohio State University Department of Molecular Genetics)
Abstract:
tRNAs undergo numerous post-transcriptional steps before they can participate in translation. In eukaryotes, there are 3 nuclear-cytoplasmic trafficking steps required for full tRNA maturation. One of these is tRNA retrograde nuclear import. This process functions in tRNA quality control, as well as synthesis of the wybutosine (yW) modification that is only present on tRNAPhe. However, it is unknown whether a tRNA can undergo more than one round of retrograde nuclear import and subsequent re-export. To address this question, I am re-engineering enzymes involved in yW synthesis. For yW to be synthesized, the tRNA must first be exported out of the nucleus into the cytoplasm where its intron is spliced out and the tRNA is methylated at positions 32 and 34 by Trm7. The tRNA then undergoes retrograde nuclear import so that nucleoplasmic Trm5 will methylate position 37 of tRNAPhe to m1G. The modified tRNAPhe is then re-exported back to the cytoplasm where Tyw1, Tyw2, Tyw3, and Tyw4 sequentially further modify position 37 to yW. Therefore, yW biogenesis requires one full round of the tRNA retrograde pathway and, thus, employing an assay we recently developed to detect yW, this tRNA modification can be utilized to monitor tRNA nuclear-cytoplasmic trafficking. To determine if a tRNA is capable of more than one round of retrograde nuclear import/re-export, I am re-localizingTyw2 or Tyw3 into the nucleus. Due to the re-localization, tRNAPhe must undergo 2 rounds of retrograde nuclear import and re-export to gain yW modification. The presence of yW modified tRNAPhe will indicate this process is iterative, but lack of yW will indicate that it is limited to once per tRNA. To direct Tyw2 to the nucleus, I tagged it at the N-terminus with GFP followed by an SV40 nuclear localization signal (NLS). Unfortunately, the altered Tyw2 does not appear to be nuclear. Therefore, additional Tyw2 and Tyw3 constructs are being generated with alternative positions of the GFP and NLS sequences.
Keywords: tRNA, Nuclear Transport, Budding Yeast