Knowledge Bank

During RNA processing, transfer RNAs (tRNAs) experience a multitude of modifications. Two such modifications are the methylation of guanosine at the positions 9 and 46. In Saccharomyces cerevisiae, these modifications at positions 9 and 46 are catalyzed by the methyltransferases Trm10 and Trm8, respectively. Deletion of trm8 in S. cerevisiae in combination with any of several other tRNA modification enzyme deletions affects growth at higher temperatures, while deletion of TRM10 was shown to have a negative effect on growth of S. cerevisiae in media containing the antitumor drug 5-fluorouracil (5FU). Overexpression of only one of the 13 Trm10 substrates, tRNATrp, has been demonstrated to restore wild-type levels of growth of a trm10Δ strain in the presence of 5FU. Because it is known that certain tRNA modification enzymes modify multiple tRNA substrates but are only necessary for some substrates, we sought to investigate whether only certain substrates need the Trm8 modification, and account for the temperature sensitivity reported for a double deletion strain of Trm8 and Trm10. We hypothesize that deletion of both enzymes leads to decreased growth of S. cerevisiae in higher temperatures and with 5FU due to one or more tRNAs being hypersensitive to the loss of these modifications. To create a trm8Δtrm10Δ double deletion strain, a trm8::Nat drug cassette containing upstream and downstream sequences of TRM8 was transformed into the trm10::Kan strain. We used DNA sequencing and replica plating on media that separately contained both drugs to confirm that the resulting strains were successfully created. Drop tests were then used to assess the growth sensitivity of the double deletion strain to each stressor. These tests revealed slower growth as the temperature/concentration of 5FU increased. Then, specific tRNAs, along with an empty tRNA vector, were chosen to overexpress in the strain because they were substrates of one or both modification enzymes. tRNAGly was chosen since it is modified by Trm10, tRNACys and tRNAVal(CAC) because they are modified by Trm8, and tRNATrp and tRNAVal(AAC) because they are modified by both enzymes. Overexpressing either of the substrates tRNATrp and tRNAVal(AAC) rescued growth in 5FU and in higher temperatures, suggesting that these two tRNAs are reliant upon these two modifications for growth under these conditions. Northern blotting techniques were used to assess tRNA levels in all strains with and without overexpressed tRNAs. This approach allowed us to determine if the levels are depleted in the strain not containing overexpressed tRNA, and whether the levels are rescued in the overexpression strain where the growth phenotype was rescued, indicating that loss of the modification impacts the abundance of the tRNA. Northern blotting showed that levels of both rescuing tRNA substrates are depleted under conditions of 5FU growth stress in each strain, thus suggesting that overexpression of tRNATrp and tRNAVal(AAC) is able to rescue growth by restoring sufficient levels of the tRNA for translation. As such, these two tRNAs are likely both affected by the loss of these modifications. As highly conserved modifications across domains of life, it is important to understand the role of these two modifications. Overall, by comparing the growth of the double deletion strain in these conditions and comparing it to the overexpression of specific tRNA substrates, these studies increase our knowledge of the biological impact of the lack of these modifications.