Nuclear surveillance and degradation of hypomodified initiator tRNAMet in S. cerevisiae

Abstract

The tRNA m(1)A58 methyltransferase is composed of two subunits encoded by the essential genes TRM6 and TRM61 (formerly GCD10 and GCD14). The trm6-504 mutation results in a defective m(1)A methyltransferase (Mtase) and a temperature-sensitive growth phenotype that is attributable to the absence of m(1)A58 and consequential tRNA(i)(Met) instability. We used a genetic approach to identify the genes responsible for tRNA(i)(Met) degradation in trm6 cells. Three recessive extragenic mutations that suppress trm6-504 mutant phenotypes and restore hypomodified tRNA(i)(Met) to near normal levels were identified. The wild-type allele of one suppressor, DIS3/RRP44, encodes a 3'-5' exoribonuclease and a member of the multisubunit exosome complex. We provide evidence that a functional nuclear exosome is required for the degradation of tRNA(i)(Met) lacking m(1)A58. A second suppressor gene encodes Trf4p, a DNA polymerase (pol sigma) with poly(A) polymerase activity. Whereas deletion of TRF4 leads to stabilization of tRNA(i)(Met), overexpression of Trf4p destabilizes the hypomodified tRNA(i)(Met) in trm6 cells. The hypomodified, but not wild-type, pre-tRNA(i)(Met) accumulates as a polyadenylated species, whose abundance and length distribution both increase upon Trf4p overexpression. These data indicate that a tRNA surveillance pathway exists in yeast that requires Trf4p and the exosome for polyadenylation and degradation of hypomodified pre-tRNA(i)(Met).

Figure 1.

Mutations of sup1, sup2, or sup3 suppress the mutant phenotypes of trm6-504. TRM6, trm6-504 and representative trm6-504, sup- strains all harboring gcn2-101 were grown to saturation in YPD medium, and serial 10-fold dilutions were spotted onto YPD plates and incubated at 36°C, 16°C, and 26°C. The same strains were grown on YPD at 26°C and replica-plated to synthetic complete (SC) plates lacking histidine and supplemented with 30 mM 3-aminotriazole (3-AT) or YPD and incubated at 30°C and 26°C, respectively.

Figure 2.

Identification of SUP1⁺ and SUP2⁺ as TRF4 and RRP44, respectively. (A) Schematic representation of genomic DNA inserts present in YCp50 (pSUP1.1 and pSUP1.2) and the single-copy plasmid subclone derived from pSUP1.1 (B254). All three plasmids complemented the Cs^- and 3-AT^r phenotypes of the sup1, trm6-504, gcn2-101 strain. (B) Schematic representation of the largest genomic DNA insert that complemented sup2 Cs^- phenotype (B227) and the single-copy plasmid subclone derived from B227 (B234). Both plasmids complemented the Cs^- and 3-AT^r phenotypes of the sup1, trm6-504, gcn2-101 strain (see Materials and Methods for details). The chromosomal coordinates for all three genomic clones containing DIS3/RRP44 (B227, B228, and B229) are given in Table 1. Black boxes, ORFs encoded on the Watson strand; stippled boxes, ORFs encoded on the Crick strand; vertical lines, incomplete ORFs. (C) Growth of strains wild type, trm6-504, sup1, trm6-504, and sup2, trm6-504 all harboring gcn2-101 transformed with YCpLac33 (rows 1-3, 5), with scTRF4 (B254, row 4) or scRRP44 (B234, row 6). Cultures of each strain grown in SC-Ura and serial 10-fold dilutions were spotted onto SC-Ura and incubated at 36°C, 16°C, and 26°C (RT). The same strains were grown as patches on SC-Ura plates and replica-plated to SC-Ura, His medium supplemented with 30mM 3-AT or SC-Ura and incubated at 30°C and 26°C, respectively.

Figure 2.

Identification of SUP1⁺ and SUP2⁺ as TRF4 and RRP44, respectively. (A) Schematic representation of genomic DNA inserts present in YCp50 (pSUP1.1 and pSUP1.2) and the single-copy plasmid subclone derived from pSUP1.1 (B254). All three plasmids complemented the Cs^- and 3-AT^r phenotypes of the sup1, trm6-504, gcn2-101 strain. (B) Schematic representation of the largest genomic DNA insert that complemented sup2 Cs^- phenotype (B227) and the single-copy plasmid subclone derived from B227 (B234). Both plasmids complemented the Cs^- and 3-AT^r phenotypes of the sup1, trm6-504, gcn2-101 strain (see Materials and Methods for details). The chromosomal coordinates for all three genomic clones containing DIS3/RRP44 (B227, B228, and B229) are given in Table 1. Black boxes, ORFs encoded on the Watson strand; stippled boxes, ORFs encoded on the Crick strand; vertical lines, incomplete ORFs. (C) Growth of strains wild type, trm6-504, sup1, trm6-504, and sup2, trm6-504 all harboring gcn2-101 transformed with YCpLac33 (rows 1-3, 5), with scTRF4 (B254, row 4) or scRRP44 (B234, row 6). Cultures of each strain grown in SC-Ura and serial 10-fold dilutions were spotted onto SC-Ura and incubated at 36°C, 16°C, and 26°C (RT). The same strains were grown as patches on SC-Ura plates and replica-plated to SC-Ura, His medium supplemented with 30mM 3-AT or SC-Ura and incubated at 30°C and 26°C, respectively.

Figure 3.

Mutations in TRF4, RRP44, and sup3 increase the steady-state level of tRNA_i^Met in a trm6-504 background. Northern blot analysis of total RNA isolated from wild-type, trm6-504, or trm6-504 trf4-20, rrp44-20 or sup3 double mutants grown at a permissive (26°C) or nonpermissive (36°C) temperature. Total RNA (10 μg) was separated by electrophoresis on a 6% denaturing acrylamide (19:1) gel cast in 8 M urea. Hybridization with probes JA11 (tRNA_i^Met), JA99 (5S rRNA), and JA151 (tRNA_CAA^Leu) were performed as described in Materials and Methods. tRNA_i^Met was visualized by autoradiography and quantified by Phosphor-Imager analysis using Image Quant software; normalized to 5S rRNA or tRNA_CAA^Leu and then expressed as a percentage of tRNA_i^Met in wild type at the same temperature. Normalization of tRNA_i^Met to 5S rRNA or tRNA_CAA^Leu yielded similar results, and the quantity of tRNA_i^Met shown is after normalizing to 5S rRNA.

Figure 4.

(A) Degradation of hypomodified tRNA_i^Met occurs in the nucleus. Wild-type (Y200), trm6-504 (Y190), rrp6Δ (F23), and trm6-50 rrp6Δ (Y298) strains were transformed with YCpLac33 (B181, rows 1-3, 5) or ScRRP6 (B269, rows 4,6), or untransformed strains ski2Δ (F24) and trm6-504 ski2Δ (Y299) were tested for growth. Transformants or ski2Δ (F24) and trm6-504 ski2Δ (Y299) were grown to saturation in SC-Ura or YPD, respectively, and serial 10-fold dilutions were spotted on SC-Ura or YPD plates and incubated at 26°C and 36°C. (B) Northern blot analysis of total RNA (10μg) isolated from wild-type (F27), trm6-504 (Y190), trm6-504 rrp6Δ (Y298), and trm6-504 ski2Δ (Y299) strains grown in YPD at permissive (26°C) and after shift to the nonpermissive temperature, 36°C. RNAs separated on a 6% polyacrylamide (19:1) 8 M urea gel and transferred to a membrane were probed with JA11 to detect tRNA_i^Met and JA151 to detect tRNA_CAA^Leu. tRNA_i^Met and tRNA_CAA^Leu were visualized by autoradiography and quantified by PhosphorImager analysis using Image Quant software. tRNA_i^Met was normalized to the amount of tRNA_CAA^Leu in the same sample and is expressed as percentage of wild type at the same temperature.

Figure 5.

Overexpression of Trf4p enhances degradation of hypomodified tRNA_i^Met in an exosome-dependent manner. (A) Wild type (Y200), trm6-504 (Y190), and trm6-504 rrp44-20 were transformed with a high-copy-number plasmid YEpLac195 (B184) or a high-copy-number plasmid bearing TRF4 (B256), and single transformants were streaked to SC-Ura and incubated for 4 d at 30°C. (B) Northern blot analysis of total RNA (10μg) isolated from the same strains described in A grown at 30°C. Detection of precursor and mature tRNA_i^Met was done by hybridization with probes JA11 (tRNA_i^Met) and JA151 (tRNA_CAA^Leu), followed by autoradiography or PhosphorImager analysis and quantification using Image Quant software. tRNA_i^Met was normalized to the amount of tRNA_CAA^Leu in the same sample and is expressed as percentage of wild type bearing the same plasmid.

Figure 6.

Pre-tRNA_i^Met lacking m¹A58 is polyadenylated. RNA isolated from the indicated strains cultured at 30°C was subjected to Northern blot analysis using total RNA (lanes 1-4, 5 μg; lane 10, 1 μg) or poly(A)⁺ RNA (lanes 5,6, 1 μg; lanes 7-9, 2 μg; lanes 11-13, 1 μg) after separation by denaturing polyacrylamide gel electrophoresis and transfer to a membrane. Hybridization with probe JA11 (tRNA_i^Met) was done as described in Materials and Methods with the exception that the hybridizing temperature was 60°C, and the tRNA_i^Met was visualized by autoradiography. Pretreatment of 1 μg of poly(A)⁺ RNA from the trm6-504 rrp6Δ +hcTRF4 strain with oligo-d(T₁₈) and RNaseH was done as described in Materials and Methods; the reactions were terminated after 1 or 3 h (lanes 12,13, respectively). The standards used in size determination were radiolabeled pBR322 digested with MspI.