Rapid tRNA Decay Can Result from Lack of Nonessential Modifications Andrei Alexandrov, Irina Chernyakov, Weifeng Gu, Shawna L. Hiley, Timothy R. Hughes, Elizabeth J. Grayhack, Eric M. Phizicky  Molecular Cell  Volume 21, Issue 1, Pages 87-96 (January 2006) DOI: 10.1016/j.molcel.2005.10.036 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Schematic of Known Locations of tRNA Modifications Catalyzed by Genes Analyzed in This Work Although all target sites of these genes are shown in the figure, different tRNAs have different sets of modifications. The modifications in tRNAVal(AAC) that are catalyzed by Trm8p/Trm82p, Trm4p, Pus7p, and Dus3p (7-methylguanosine [m7G46], 5-methylcytidine [m5C49], pseudouridine [Ψ13], and dihydrouridine [D47], respectively) are indicated by filled circles and discussed in detail below. Modifications that are not shown but are described in the text include: N6-isopentenyladenosine (i6A), 2′-O-ribosyladenosine phosphate (A[r]), 1-methyladenosine (m1A), inosine (I), 1-methylinosine (m1I), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-carbamoylmethyluridine (ncm5U), 5-methyluridine (m5U), 1-methylguanosine (m1G), N2,N2-dimethylguanosine (m22G), 2′-O-methylguanosine (Gm), N2-methylguanosine (m2G), and 2′-O-methylnucleoside (2′-O-Me). Molecular Cell 2006 21, 87-96DOI: (10.1016/j.molcel.2005.10.036) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Mutants Lacking Certain Combinations of tRNA Modifying Enzymes Are Temperature Sensitive on YPD Media Diploid strains, bearing all possible combinations of homozygous double-deletion mutations of indicated genes, were constructed individually as described in Experimental Procedures (strain genotypes are shown in Table S3). Overnight cultures were diluted and spotted on YPD plates (5,000–10,000 cells per spot) and incubated as indicated. (A) Growth at 37°C. (B) Growth at 28°C. The control strains included are: diploid strains lacking both an indicated tRNA modifying gene and a control gene, YNL063w (row j), and diploid strains lacking only one gene (row k). No cells were spotted along the diagonal (from a1 to j10), except for the wild-type strain at the intersection of row k and column 11. (C) Complementation of the growth defect with single-copy plasmids containing respective missing genes. Each strain shown in (A) that was found to be defective for growth was transformed with a single-copy plasmid containing the appropriate gene under control of its own promoter as indicated, and cultures were diluted as described above and tested for growth on SD-leu at 37°C and 28°C. Molecular Cell 2006 21, 87-96DOI: (10.1016/j.molcel.2005.10.036) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Levels of tRNAVal(AAC) Are Reduced in Double-Mutant Strains and Decrease Rapidly in a trm8-Δ trm4-Δ Strain (A) Northern blot analysis of tRNAVal(AAC) in mutant strains. Wild-type and mutant strains were grown at 28°C and either continued at this temperature or shifted to 37°C for 3 hr, and RNA was isolated and analyzed by Northern blotting as described in the Experimental Procedures. (B) Quantification of the levels of tRNAVal(AAC) from the Northern blot shown in (A). The ordinate shows the ratio of the levels of tRNAVal(AAC) at 37°C to that at 28°C (each value itself first normalized to 5S RNA). (C) Levels of tRNAVal(AAC) decrease rapidly after the temperature shift. Wild-type and trm8-Δ trm4-Δ double mutants were grown at 28°C and shifted to 37°C for the indicated times, and RNA was analyzed by Northern blotting. (D) Quantification of steady-state levels from the Northern blot shown in (C). The ordinate shows the ratio of the levels of the indicated tRNA species to the levels of the same tRNA species in the wild-type strain immediately before temperature shift (each value itself first normalized to 5S RNA). Molecular Cell 2006 21, 87-96DOI: (10.1016/j.molcel.2005.10.036) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Loss of Aminoacylation Specific for tRNAVal(AAC) Is Observed in trm8-Δ trm4-Δ Strains at 37°C Wild-type, trm8-Δ, trm4-Δ, and trm8-Δ trm4-Δ mutants were grown at 28°C and shifted to 37°C for the indicated times, as described in the Experimental Procedures. Then, total RNA was isolated under acid conditions to preserve aminoacylation, and 20 μg samples were resolved on 6.5% acid gels and analyzed by Northern blotting for aminoacylation. Control samples were treated with base to remove the amino acid prior to polyacrylamide gel electrophoresis (PAGE). Upper and lower arrows in each section indicate positions of aminoacylated and de-aminoacylated tRNA species. Shown below is the % aminoacylation for tRNAVal(AAC). Molecular Cell 2006 21, 87-96DOI: (10.1016/j.molcel.2005.10.036) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Complementation of Growth Defect by tRNAVal(AAC) Introduced on a Multicopy Plasmid Wild-type, trm8-Δ, trm4-Δ, and trm8-Δ trm4-Δ mutants were transformed with a multicopy plasmid, yEPlac195, containing tRNAVal(AAC) or a vector control, and transformants were grown overnight at 28°C in SD-ura liquid media, were serially 10-fold diluted, spotted on YPD plates, and incubated at different temperatures (30°C, 33°C, or 37°C) as indicated. Molecular Cell 2006 21, 87-96DOI: (10.1016/j.molcel.2005.10.036) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Rapid Degradation of tRNAVal(AAC) in trm8-Δ trm4-Δ Mutants at 37°C Does Not Require TRF4, TRF5, SKI2, or RRP6 (A) tRNA levels in trm8-Δ trm4-Δ mutants and corresponding trf4-Δ and rrp6-Δ triple mutants. Strains were grown at 28°C and shifted to 37°C for the indicated times, and then total RNA was isolated and analyzed by Northern blot as described in the Experimental Procedures. (B) Quantification of steady-state levels of tRNAVal(AAC) from Northern blots shown in (A). The ordinate shows the ratio of the levels of tRNAVal(AAC) in indicated strains to the levels in the wild-type strain immediately before temperature shift (each value itself first normalized to 5S RNA). Quantification of tRNAArg(ACG) and tRNAGlu(TTC) is shown in Figure S4. (C) tRNA levels in control strains. trm8-Δ trm4-Δ strains, their corresponding trf4-Δ, trf5-Δ, ski2-Δ, and rrp6-Δ triple mutants, and control single mutants were grown at 28°C and shifted to 37°C for 25 min, and total RNA was analyzed by Northern blot as described in the Experimental Procedures. Quantification of steady-state levels of tRNAVal(AAC) is shown at the bottom of the panel. Levels of tRNAVal(AAC) were normalized to 5S RNA and then to that in the wild-type strain immediately before temperature shift. Molecular Cell 2006 21, 87-96DOI: (10.1016/j.molcel.2005.10.036) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 Deletion of TRF4 or RRP6 Partially Rescues the Growth Defect and the Loss of tRNAVal(AAC) of trm8-Δ trm4-Δ Strains at 33°C, but Not at 37°C (A) Complementation of the growth defect of trm8-Δ trm4-Δ mutants at 33°C by deletion of TRF4 or RRP6. Strains, as indicated, were grown overnight and serial 10-fold dilutions were plated on YPD media (starting from 20,000 cells) and incubated at 30°C, 33°C, and 37°C as indicated. (B) Deletion of TRF4 or RRP6 increases levels of tRNAVal(AAC) in a trm8-Δ trm4-Δ strain at 33°C. trm8-Δ trm4-Δ mutants and corresponding trf4-Δ and rrp6-Δ triple mutants were grown at 28°C and shifted to 33°C for 13 hr, and then RNA was isolated and analyzed by Northern blot, as described in the Experimental Procedures. Steady-state levels of tRNAVal(AAC) were normalized to 5S RNA and then to that in the trm8-Δ trm4-Δ strain before temperature shift (which is itself set to 60%, as determined above in Figure 3, [C] and [D] and Figure 6, [A] and [B]). Molecular Cell 2006 21, 87-96DOI: (10.1016/j.molcel.2005.10.036) Copyright © 2006 Elsevier Inc. Terms and Conditions