Rex1p deficiency leads to accumulation of precursor initiator tRNAMet and polyadenylation of substrate RNAs in Saccharomyces cerevisiae

Abstract

A synthetic genetic array was used to identify lethal and slow-growth phenotypes produced when a mutation in TRM6, which encodes a tRNA modification enzyme subunit, was combined with the deletion of any non-essential gene in Saccharomyces cerevisiae. We found that deletion of the REX1 gene resulted in a slow-growth phenotype in the trm6-504 strain. Previously, REX1 was shown to be involved in processing the 3' ends of 5S rRNA and the dimeric tRNA(Arg)-tRNA(Asp). In this study, we have discovered a requirement for Rex1p in processing the 3' end of tRNA(i)(Met) precursors and show that precursor tRNA(i)(Met) accumulates in a trm6-504 rex1Delta strain. Loss of Rex1p results in polyadenylation of its substrates, including tRNA(i)(Met), suggesting that defects in 3' end processing can activate the nuclear surveillance pathway. Finally, purified Rex1p displays Mg(2+)-dependent ribonuclease activity in vitro, and the enzyme is inactivated by mutation of two highly conserved amino acids.

Figure 1.

A trm6-504 rex1Δ double mutant strain displays a synthetic slow-growth phenotype. Wt (Y200), trm6-504 (Y190), trm6-504 rex1Δ (Y386) and rex1Δ (Y387) yeast strains were streaked to SD^+His+Ura and grown at room temperature for 3 days.

Figure 2.

A trm6-504 rex1Δ strain accumulates tRNA_i^Met encoded by IMT2 and IMT3. (A) Total RNA (5 μg) isolated from Wt (Y200), trm6-504 (Y190), trm6-504 rex1Δ (Y386) and rex1Δ (Y387) strains was separated by electrophoresis using an 8% denaturing polyacrylamide gel. Northern analysis was performed with radiolabeled oligonucleotides that hybridize to tRNA_i^Met (JA11) or 5S rRNA (JA99). Results were visualized by autoradiography. (B) Northern analysis of total RNA (10 μg) isolated from a trm6-504 strain (H2457) carrying one of the four IMT genes on a high-copy plasmid. The blot was probed for tRNA_i^Met (JA11) and the results were visualized by autoradiography. (C) An alignment of the tRNAs_i^Met encoded by the four IMT genes from S. cerevisiae. A portion of the sequence found in the mature tRNA is shown in a black background. The 3′ trailer sequences are shown in black text, with the putative RNA Polymerase III termination sites underlined. A downstream terminator IMT4 is underlined with a dashed line. The imt4-3 mutant has an alteration in the length of the 3′ trailer preceding the first transcriptional terminator. (D) Northern analysis of total RNA (20 μg) performed as described in (A), probing with oligonucleotides complementary to the 5′ leader of IMT2- or IMT3-encoded tRNA_i^Met (JA72 or JA48, respectively).

Figure 3.

Changes in the 3′ trailer length influence tRNA_i^Met accumulation in a trm6-504 rex1Δ strain. (A) Wt (Y200) or trm6-504 (Y190) strains carrying vector (YCplac33), hcIMT4, or hcimt4-3 were grown at 30°C for 2 days on SC^−URA. (B) Total RNA (20 μg) isolated from Wt (Y200), trm6-504 (Y190), trm6-504 rex1Δ (Y386) and rex1Δ (Y387) strains carrying empty vector (YCplac33), hcIMT4 (pJA108) or hcimt4-3 (p467) was subjected to Northern analysis. The blot was probed with a radiolabeled oligonucleotide complementary to the 5′ leader of IMT4 (JA66), and the results visualized using phosphorimaging. The blot was stripped, probed again with an oligonucleotide that hybridizes to 5S rRNA (JA99), and analyzed by phosphorimaging.

Figure 4.

An increase in the level of polyadenylated tRNA_i^Met is seen in a trm6-504 rex1Δ strain. (A) Total RNA (5 μg) and poly(A) + RNA (3–4 μg, normalized to the total RNA yield, see ‘Methods’ section) was separated on a 6% denaturing polyacrylamide gel. Northern analysis was performed with a radiolabeled oligonucleotide complementary to tRNA_i^Met (JA11). (B) A bar graph of the relative amounts of poly(A) + tRNA_i^Met in the Wt, trm6-504, trm6-504 rex1Δ and rex1Δ strains, as seen in (A). Quantitation was performed using ImageQuant TL software (GE Healthcare). (C) The blot in (A) was stripped and probed for tRNA^Val(CAC) (JA555). Results were visualized by autoradiography for both blots. The mature tRNA in the poly(A) + lanes represents tRNA that was not eliminated by oligo-(dT) selection.

Figure 5.

Loss of Rex1p results in polyadenylation of 5S rRNA and tRNA^Arg from the dimeric tRNA^Arg-tRNA^Asp transcript. (A) Total and poly (A) + RNA were analyzed as described for Figure 4. The blot was probed for 5S rRNA (JA99) and the results visualized by autoradiography. (B) Northern analysis was performed using a labeled oligonucleotide (JA557) that hybridizes to the 3′ end of tRNA^Arg and the linker sequence between the two tRNAs. Phosphorimaging was used to analyze results. The presence of mature forms of RNAs in the poly(A) + lanes represent RNAs not eliminated during oligo-(dT) selection.

Figure 6.

Rex1p displays tRNA 3′end processing activity in vivo and in vitro. (A) A diagram of Rex1p showing the conserved amino acids found in the three Exo motifs. An alignment of Rex1, 2, 3 and 4, and E. coli RNase T protein sequence is shown underneath to illustrate the conservation of the D × E sequence found in the ExoI domain. (B) Wt and mutant FLAG-tagged Rex1p were purified from yeast (strains Y438 and Y495, respectively) using affinity chromatography. Purified protein was subjected to SDS-PAGE and visualized by Coomassie staining. The positions of molecular weight standards (Broad Range Protein Marker, New England Bioloabs) are indicated. (C) Gel purified ³²P- 5′ end labeled tRNAs_i^Met (∼ 10 pM) were incubated in buffer alone or with Wt or mutant Rex1p (∼7.5 nM) at 30°C for 10 min. Reaction products were separated on a 10% denaturing polyacrylamide gel and visualized by autoradiography. The positions of RNAs of known length (Decade Marker, Ambion) are indicated (in nucleotides). (D) Wt Rex1p (∼7.5 nM) was incubated with labeled ‘IMT3’ tRNA_i^Met (∼20 pM) for known periods of time from 0.5–7 min, as indicated. Control reactions with ‘IMT3’ tRNA_i^Met lacking either enzyme or Mg²⁺ are shown, as well as a control reaction lacking enzyme that contained the ‘mature’ tRNA_i^Met. After separation on a denaturing 10% polyacrylamide gel, results were visualized using phosphorimaging. (E) Northern analysis of total RNA (10 μg) isolated from a trm6-504 rex1Δ strain carrying an empty vector (pRS316), or a plasmid with galactose-inducible Wt REX1 (pAV101) or mutant rex1 (p532), grown under non-inducing or inducing conditions. The blot was probed with a radiolabeled oligonucleotide complementary to tRNA_i^Met (JA11) and the results visualized using autoradiography.