Identification of the TRM2 gene encoding the tRNA(m5U54)methyltransferase of Saccharomyces cerevisiae

Abstract

The presence of 5-methyluridine (m5U) at position 54 is a ubiquitous feature of most bacterial and eukaryotic elongator tRNAs. In this study, we have identified and characterized the TRM2 gene that encodes the tRNA(m5U54)methyltransferase, responsible for the formation of this modified nucleoside in Saccharomyces cerevisiae. Transfer RNA isolated from TRM2-disrupted yeast strains does not contain the m5U54 nucleoside. Moreover, a glutathione S-transferase (GST) tagged recombinant, Trm2p, expressed in Escherichia coli displayed tRNA(m5U54)methyltransferase activity using as substrate tRNA isolated from a trm2 mutant strain, but not tRNA isolated from a TRM2 wild-type strain. In contrast to what is found for the tRNA(m5U54)methyltransferase encoding gene trmA+ in E. coli, the TRM2 gene is not essential for cell viability and a deletion strain shows no obvious phenotype. Surprisingly, we found that the TRM2 gene was previously identified as the RNC1/NUD1 gene, believed to encode the yNucR endo-exonuclease. The expression and activity of the yNucR endo-exonuclease is dependent on the RAD52 gene, and does not respond to increased gene dosage of the RNC1/NUD1 gene. In contrast, we find that the expression of a trm2-LacZ fusion and the activity of the tRNA(m5U54)methyltransferase is not regulated by the RAD52 gene and does respond on increased gene dosage of the TRM2 (RNC1/NUD1) gene. Furthermore, there was no nuclease activity associated with a GST-Trm2 recombinant protein. The purified yNucR endo-exonuclease has been reported to have an NH2-D-E-K-N-L motif, which is not found in the Trm2p. Therefore, we suggest that the yNucR endo-exonuclease is encoded by a gene other than TRM2.

Dual function of the tRNA(m5U54)methyltransferase in tRNA maturation

“…Strains with a mutation in the sup61 + gene required the TRM2 gene for growth A strain deleted for the TRM2 gene is viable and exhibits no apparent phenotype (Nordlund et al+, 2000)+ To elucidate the function of the Trm2 protein and/or the m 5 U 54 nucleoside in tRNA, a synthetic (synergistic) lethal screen was performed employing a red/white colony color assay (Kranz & Holm, 1990;Bender & Pringle, 1991)+ Synergistic lethality provides genetic evidence that two components physically interact or have overlapping functions+ Briefly, a haploid ura3 ade2 ade3 trm2 strain carrying a plasmid with the ADE3, URA3, and TRM2 genes was mutagenized and colonies were screened for the inability to lose the plasmid that is scored as uniformly red (nonsectored) colonies+ From 90,000 colonies, 21 strains with recessive mutations were identified that required the TRM2 gene for survival (see Materials and Methods)+ The mutants fell into several complementation groups of which one with four mutants was studied further+ The nonsectoring phenotype of these four strains was linked to a temperaturesensitive (Ts) growth phenotype+ Using a yeast genomic library, we identified three separate plasmids with identical inserts, which fully complemented the Ts and nonsectoring phenotypes+ Partial DNA sequencing of the insert from one of these plasmids and subsequent subcloning revealed that the sup61 ϩ gene restored sectoring at 30 8C and growth at 37 8C+ We also isolated plasmids carrying the eEF1A (TEF2) and the seryl-tRNA synthetase (SES1) genes that were able to partially complement the mutants+ Apparently, the TEF2 and SES1 genes were acting as dosage suppressors of the mutant phenotype (our unpubl+ data)+ The sup61 ϩ gene is a single copy and essential gene encoding tRNA CGA Ser , which is the only serine isoacceptor that decodes UCG codons (Etcheverry et al+, 1982)+ To confirm that sup61 ϩ was mutated, we cloned and sequenced the gene from the four mutant strains+ This analysis revealed that each strain contained a different single nucleotide exchange in the sup61 gene and the alleles were designated sup61-T51C, sup61-T44G, sup61-T47:2C, and sup61-T20A (Fig+ 1A)+ To unambiguously demonstrate that the various sup61 alleles were responsible for the dependency on TRM2, the wild-type sup61 ϩ gene was replaced with respective mutant allele in a trm2 strain carrying TRM2 on a plasmid (see Materials and Methods)+ The resulting strains (UMY2244 to 2247) exhibited a Ts growth phenotype and were dependent on the TRM2 plasmid for growth at 30 8C+ At 25 8C, the strains were able to spontaneously lose the TRM2 plasmid and generate the double mutant strains+ These double mutants did not to grow (sup61-T44G trm2, sup61-T20A trm2) or grew very slowly (sup61-T51C trm2, sup61-T47:2C trm2) at 30 8C, whereas the single trm2 and sup61 strains were able to grow (Fig+ 1B)+ Thus, the m 5 U modification or the Trm2 protein is important for growth in strains with mutant forms of tRNA CGA Ser + The Trm2 gene product influenced the relative level of mature and pre-tRNA CGA Ser in the sup61 mutants To establish the molecular mechanism for the synergistic effect of the trm2 null and the various sup61 alleles, the relative amount of tRNA CGA Ser was determined using northern blot analysis+ Wild-type, single, and double mutant strains were grown exponentially at 25 8C and shifted to 30 8C for 4 h, and total RNA was prepared from samples taken at respective temperature+ Because the sup61 gene contains an intervening sequence (Etcheverry et al+, 1979), mature and pre-tRNA CGA Ser was detected by using two different 32 Plabeled oligonucleotides as probes+ One probe was complementary ...…”

“…Total RNA for northern blots was prepared using glass beads as described (Ausubel et al+, 2001) Figure 6+ To detect tRNA i Met , the oligonucleotide 59-GGACATCAGGGTTATGA GCC-39 was used+ Oligonucleotides were labeled using adenosine 59[g 32 P]-triphosphate (5,000 Ci/mmol, Amersham) and polynucleotide kinase (Roche Molecular Biochemicals)+ Northern blots were visualized and quantified by PhosphorImager analysis+ Total tRNA for HPLC analysis was prepared essentially as described (Nordlund et al+, 2000) except for the 5-min vortexing step, which was shortened to 2 min+ The tRNA elution buffer was 0+8 M NaCl, 50 mM Tris-HCl, pH 8+0, 15% ethanol+ For HPLC analysis, 50 mg tRNA were digested to nucleosides using nuclease P1 and bacterial alkaline phosphatase (Gehrke et al+, 1982) and the hydrolysate was analyzed by HPLC (Gehrke & Kuo, 1990) using a Waters TM System liquid chromatograph with a Waters TM 996 diode array UV detector (Waters TM Corporation, Milford, Massachusetts, USA)+ Separation of nucleosides was achieved using a Supelcosil LC-18-S reverse-phase column (4+6 by 250 mm) and a Supelguard LC-18-S, 2+1 by 20 mm guard column (Supelco, Bellefonte, Pennsylvania, USA) thermostatted to 26 8C, at a flow rate of 1 mL/min+ For MS analysis, the flow rate was 2 mL/min, and the UV detector was directly interfaced to a VG platform mass spectrometer equipped with an electrospray ionization source (Fisons Instruments, Altrincham, UK)+ In MS analysis, nucleosides were eluted using the gradient of Buck et al+ (1983)+ UV data were recorded continuously, and mass spectra were recorded every 1+0 s during the 60-min chromatography+ The procedures and interpretation of data for qualitative LC-MS analysis of nucleosides in RNA hydrolysates have been previously described in detail (Pomerantz & McCloskey, 1990)+…”

“…The source and genotypes of yeast strains used in this study are listed in Table 2+ E. coli strains used were DH5a (Bethesda Research Laboratories) and CJ236 (Kunkel et al+, 1987)+ Yeast transformation (Gietz et al+, 1992), media, and genetic procedures have been described (Rose et al+, 1990)+ Strain UMY1698 was generated from CH1305 using plasmid YRp14/ trp1⌬1 (Sikorski & Hieter, 1989)+ Strains UMY1917, UMY2097, and UMY2228 were constructed by replacing the TRM2 gene with a trm2::LEU2 construct in UMY1698, UPX16-11C, and UMY2219 as described previously (Nordlund et al+, 2000)+ Disruption of TRM2 was confirmed by PCR combined with HPLC analysis of nucleosides derived from total tRNA isolated from the disrupted strain+ Strain UMY2380 was constructed by replacing the LHP1 sequence between position Ϫ10 and 710, relative to the A residue in the AUG codon, in strain UMY2220 with a functional URA3 gene+ This was done by PCR-mediated gene disruption (Brachmann et al+, 1998) using oligonucleotides 59-ATGGGTTCTATTTGGTTCTACTG GAACTAAAGTAGCATCTAGATTGTACTGAGAGTGCAC-39 and 59-AAAGGCTTCCAAGTTCTCCTGCAATTCAGGAAT TTGGGAACTGTGCGGTATTTCACACCG-39+ Strains UMY 2527, UMY2526, and UMY2592 were constructed in a similar fashion, replacing the entire PUS4, TRM1, and TRM3 open reading frames with functional TRP1 or URA3 genes+ Disruptions of LHP1, PUS4, TRM1, and TRM3 were confirmed by PCR+ The wild-type sup61 ϩ gene was replaced with mutant alleles in strain UMY2241 (UMY2228 carrying pMJ1005) using the two-step method (Scherer & Davis, 1979)+ To target the mutant sup61 alleles to the correct chromosomal location (Orr-Weaver et al+, 1981) a unique Bgl II site was utilized to linearize the pRS306-sup61 vectors (pMJ1230-1233)+ After integration and selection for 5-FOA R segregants, colonies were screened for a nonsectoring phenotype+ Strains UMY2244, UMY2245, UMY2246, and UMY2247 were isolated by this approach and confirmed by the ability sector when transformed with pRS316-TRM2+ These strains were able to spontaneously lose the TRM2 plasmid at 25 8C generating UMY2258, UMY2259, UMY2260, and UMY2261+ Strains with mutant sup61 alleles were obtained from tetrads from crosses between UMY2220 and UMY2244, UMY2245, UMY2246, and UMY2247+ The progeny showed a 2:2 segregation of the Ts phenotype and strains UMY2254, UMY2255, UMY2256, and UMY2257 were derived from Ts, Leu Ϫ and Trp Ϫ spore colonies+ These strains were confirmed by complementing the Ts phenotype with pRS316-sup61 ϩ + The sup61 lhp1, sup61 pus4, sup61 trm1, and sup61 trm3 strains were obtained from tetrads from crosses between UMY2380, UMY2527, UMY2526, UMY2592, and strains carrying the mutant sup61 alleles (UMY2254-2257)+ Synthetic lethal screen and cloning of the sup61 1 gene…”

“…DNA manipulations, plasmid preparations, and bacterial transformations were performed according to standard protocols (Sambrook et al+, 1989)+ Plasmid pMJ1223 was constructed by replacing the Sal I spADH::CLN2 fragment in plasmid c2013 (Cvrckova & Nasmyth, 1993) with a Sal I RHO4-TRM2 fragment from pMJ1342 that is pRS316-RHO4-TRM2 (Nordlund et al+, 2000) with an additional Sal I site introduced at the EclXI site in the cassette+ The TRP1 variant of the ADE3 TRM2 plasmid (pMJ1005) was created by transforming a yeast strain carrying pMJ1223 with a ura3::TRP1 construct+ The plasmid was isolated from a Trp ϩ Ura Ϫ strain and confirmed by restriction analysis+ A low copy TRP1 vector carrying the TRM2 gene (pMJ1274) was constructed by cloning a Bgl II/EclXI TRM2 fragment from pRS316-RHO4-TRM2 into the BamHI/EclXI sites of pRS414 (Sikorski & Hieter, 1989)+ Mutant alleles of TRM2 were obtained by oligo-directed mutagenesis on pRS316-TRM2 (Nordlund et al+, 2000) according to Kunkel et al+ (1987) for pRS316-trm2-C521A (pMJ1248) and using the QuickChange TM site-directed mutagenesis kit (Stratagene) for pRS316-trm2-G428D (pMJ1195)+ The G428D exchange was based on the mutational analysis of the AdoMet binding site in the EcoKI N6-adenine methyltransferase (Willcock et al+, 1994)+ Integrative vectors carrying mutant sup61 genes were constructed by cloning a HindIII/EcoRI fragment PCR amplified from respective mutant strain into the corresponding sites of pRS306 (Sikorski & Hieter, 1989)+ This generated pRS306-sup61-T51C (pMJ1230), pRS306-sup61-T44G (pMJ1231), pRS306-sup61-T47:2C (pMJ1232), and pRS306-sup61-T20A (pMJ1233)+ The oligonucleotides used for the PCR were 59-GAGTGAGAAGCTTATATGC-39 and 59-TTGAATTCTGTGCTAAGTCTGTGAC-39+ The wildtype sup61 ϩ gene was amplified from UMY1917 using the same oligonucleotides as above and cloned into pRS316 (Sikorski & Hieter, 1989) generating pRS316-sup61 ϩ (pMJ1271)+…”

“…Eukaryotic transfer RNA genes are transcribed by RNA polymerase III generating precursor tRNAs that have to be further processed to generate functional tRNAs (for review, see Wolin & Matera, 1999)+ One of the first proteins that bind the newly synthesized pre-tRNA is the La protein (Lhp1p in yeast), which binds to the 39 end of the transcript and protects this end from exonucleolytic digestion (Yoo & Wolin, 1997;Fan et al+, 1998)+ This is followed by removal of the 59 leader by RNase P (for review, see Frank & Pace, 1998), which precedes an endonucleolytic removal of the 39 trailer+ Furthermore, eukaryotic tRNAs have to undergo posttranscriptional addition of CCA to their 39 termini catalyzed by the tRNA nucleotidyltransferase (for review, see Deutscher, 1990)+ Some tRNA genes contain an intron and splicing of the pre-tRNA occurs before or after end maturation+ However, in Saccharomyces cerevisiae, end maturation normally precedes splicing (O'Connor & Peebles, 1991)+ During the maturation of tRNA, a variety of different nucleoside modifications occurs that allows the mature tRNA to function with high efficiency and flexibility+ Modified nucleosides can be found in all phylogenetic domains and also in identical positions of the tRNA, suggesting a conserved function of some tRNA modifications (Björk, 1986;Cermakian & Cedergren, 1998;Björk et al+, 2001)+ A 5-methyluridine residue at position 54 (m 5 U 54 ) is a highly conserved feature of eukaryotic and bacterial tRNAs+ The presence of this modification influences, in vitro, the fidelity and rate of protein synthesis as well as the stability of tRNA tertiary structure (Davanloo et al+, 1979;Kersten et al+, 1981)+ The in vivo role has been more difficult to elucidate+ An Escherichia coli strain with a mutation in the gene (trmA) encoding the tRNA(m 5 U 54 )methyltransferase and that lacks the m 5 U 54 nucleoside show a slight reduction in growth rate (Björk, 1986)+ However, a truncation of the trmA gene was shown to be lethal, indicative of an additional and essential function of the protein (Persson et al+, 1992)+ In contrast, an S. cerevisiae strain with either a mutation in or a deletion of the TRM2 gene, encoding the yeast tRNA(m 5 U 54 )methyltransferase, lacks the m 5 U 54 nucleoside but is viable and exhibits no apparent phenotype (Hopper et al+, 1982;Nordlund et al+, 2000)+ The methylation of U 54 is an early event in tRNA maturation in S. cerevisiae, as intron-containing pre-tRNAs have been shown to contain the m 5 U nucleoside (Knapp et al+, 1978;Etcheverry et al+, 1979)+ In this study, we report that yeast strains with each of four different mutant alleles of the single copy and essential sup61 ϩ gene, encoding tRNA CGA Ser , have a requirement for the TRM2 gene for growth at 30 8C+ The absence of the Trm2 protein ...…”