Trm112p is a 15-kDa zinc finger protein essential for the activity of two tRNA and one protein methyltransferases in yeast

Abstract

The degenerate base at position 34 of the tRNA anticodon is the target of numerous modification enzymes. In Saccharomyces cerevisiae, five tRNAs exhibit a complex modification of uridine 34 (mcm(5)U(34) and mcm(5)s(2)U(34)), the formation of which requires at least 25 different proteins. The addition of the last methyl group is catalyzed by the methyltransferase Trm9p. Trm9p interacts with Trm112p, a 15-kDa protein with a zinc finger domain. Trm112p is essential for the activity of Trm11p, another tRNA methyltransferase, and for Mtq2p, an enzyme that methylates the translation termination factor eRF1/Sup45. Here, we report that Trm112p is required in vivo for the formation of mcm(5)U(34) and mcm(5)s(2)U(34). When produced in Escherichia coli, Trm112p forms a complex with Trm9p, which renders the latter soluble. This recombinant complex catalyzes the formation of mcm(5)U(34) on tRNA in vitro but not mcm(5)s(2)U(34). An mtq2-0 trm9-0 strain exhibits a synthetic growth defect, thus revealing the existence of an unexpected link between tRNA anticodon modification and termination of translation. Trm112p is associated with other partners involved in ribosome biogenesis and chromatin remodeling, suggesting that it has additional roles in the cell.

FIGURE 1.

Co-expression of Trm9p and Trm112p in E. coli. Extracts were prepared from an E. coli strain transformed with a plasmid expressing Trm9p either alone (lanes 1 and 2) or with Trm112p (lanes 3 and 4). Lanes 1 and 3, insoluble pellet (P); lanes 2 and 4, soluble fraction (S). Western blot analysis of S-tagged Trm9p (top panels) and His₆-tagged Trm112p (bottom panels) is shown. Then a total extract (lane 5, T) was purified on a nickel-nitrilotriacetic acid column, and after extensive washes, the retained proteins were eluted with 250 mm imidazole (lane 6, E). Lanes 5 and 6, Coomassie Blue staining.

FIGURE 2.

HPLC chromatograms recorded at A_{254 nm} (left panels) and A_{314 nm} (right panels). tRNA was degraded to nucleosides and separated on a C30 reverse phase column. The positions of relevant modified nucleosides are indicated. Arrow a, cm⁵U; arrow b, ncm⁵U; arrow c, mcm⁵U; arrow d, m¹G; arrow e, m²G; arrow f, mcm⁵s²U; arrow g, cm⁵s²U; arrow h, ncm⁵s²U. wt, wild type.

FIGURE 3.

HPLC analysis of in vitro modified nucleosides. tRNA prepared from a trm9-0 strain was treated or not with 5 μg of recombinant Trm9p/Trm112p enzyme. Then it was degraded and analyzed as in Fig. 2. The arrow in each panel points to the peak of mcm⁵U, which is detected only after incubation with recombinant Trm9p/Trm112p (bottom panel).

FIGURE 4.

Growth-rate comparison of wild-type and mutant strains. A trm9-0 mtq2-0 double mutant strain exhibits a synthetic growth defect, as compared with the two single mutant parent strains (W303 background). A, A_{600 nm} of various cultures grown in YPD at 30 °C were plotted at different times, as indicated. The generation time was calculated for each strain as the mean value of three independent experiments: wild type (WT, BMA64–1A), 90 ± 5 min; trm9 (YBL4557), 120 ± 5 min; mtq2 (YBL4731), 150 ± 5 min; trm9 mtq2 (YBL4740), 210 ± 5 min; trm112 (YBL4663), 360 ± 10 min. B, a diploid strain heterozygous for trm9-0 and for mtq2-0 was sporulated, and the tetrads thus obtained were dissected onto YPD plates that were incubated at 30 °C for various period of times. The pictures were taken, and then the colonies were tested for their genetic markers and by PCR analysis to test for the presence of the wild-type or the deleted alleles. The results for two independent tetrads (lanes a and b) are shown, the four resulting spores being shown vertically (rows 1–4). The results of the genotypic analysis for each spore are shown on both sides of the figure: on the left for tetrad a and on the right for tetrad b. The results are comparable with those of the liquid cultures shown above; the mtq2-0 spores (lane b, rows 2 and 3) grew more slowly than the trm9-0 (lane b, rows 1 and 4), which themselves grew more slowly than the wild type (lane a, rows 2 and 4). On the other hand, the two double mutant spores (lane a, rows 1 and 3) grew significantly more slowly than each single mutant parent. See also supplemental Table S3.

FIGURE 5.

tRNA steady-state levels in different mutants. A, total RNA was separated on denaturing 1.2% agarose gel, transferred under vacuum to charged nylon membrane, and probed as indicated. K, D, and G, probes for tRNA Lys, Glu, and Gly, respectively. scR1, the three membranes were reprobed with scR1 for normalization. The origin of each RNA sample is shown at the top; the five lanes on the left are from strains constructed in the W303 background, and the four lanes on the right, with asterisks, are in the S288c background. wt, wild type. B, a diploid strain heterozygous for the trm112-0 deletion was transformed with high copy number plasmids expressing tRNA^Lys, tRNA^Glu, tRNA^Gln, or the three tRNA together (EKQ), as indicated. These strains were then sporulated, the tetrads were dissected, and the growth of the resulting spores containing the plasmids was tested at 30 °C. Shown here are the results for two tetrads (lanes a and b) for each transformant. The large colonies (such as lane a, rows 1 and 4, and lane b, rows 1 and 3, for tRNA^Lys, for instance) were tested and found to be wild type for TRM112, whereas the small ones were trm112::kanMX4. None of the tRNA expressing plasmids was able to compensate for the growth defect of the trm112-0 strain.

FIGURE 6.

Partners of Trm112p. The figure schematically represents six putative partners of Trm112p. The four proteins represented in light shading correspond to the ones that were predicted to be Trm112p partners when we initiated this study (10, 34) and that were then confirmed individually by co-immunoprecipitation. They all possess a Rossman fold domain that is the signature of enzymes utilizing S-adenosylmethionine as a co-factor, and they are all confirmed MTases, except for Lys9p, which lacks one of the seven β-strands of the fold. The proteins shown in darker shading were reported more recently and do not possess a Rossman fold. Ecm16/Dhr1p was shown to interact with Trm112p in a recent proteomic analysis (52). It is a DEAH-box ATP-dependent RNA helicase involved in rRNA maturation (53). Sfh1p was then reported to interact with Trm112p in a two-hybrid interactome network (36). It is a component of the RSC complex that is phosphorylated during the G₁ phase of the cell cycle. It is encoded by an essential gene, and the protein is required for cell cycle progression and maintenance of proper ploidy (37, 38). Two more proteins are represented on a white background, with arrows pointing to TRM112 mRNA. Hek2/Khd1p and Whi3p are RNA-binding proteins that were found to participate in the localization of numerous mRNA at the tip of the bud (54) and at the endoplasmic reticulum (55), respectively.

FIGURE 7.

Nuclear genome instability in WT versus trm112-0 mutant. The values represent the rates of CAN 5-fluoro-orotic acid-resistant clones/million cells (see “Experimental Procedures” and Ref. 27), indicative of chromosome break or loss of the distal arm on chromosome V. CIN, chromosome instability; WT, wild type.