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. 2008 Jan;14(1):107-16.
doi: 10.1261/rna.808608. Epub 2007 Nov 13.

Degradation of Hypomodified tRNA(iMet) in Vivo Involves RNA-dependent ATPase Activity of the DExH Helicase Mtr4p

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Free PMC article

Degradation of Hypomodified tRNA(iMet) in Vivo Involves RNA-dependent ATPase Activity of the DExH Helicase Mtr4p

Xuying Wang et al. RNA. .
Free PMC article

Abstract

Effective turnover of many incorrectly processed RNAs in yeast, including hypomodified tRNA(iMet), requires the TRAMP complex, which appends a short poly(A) tail to RNA designated for decay. The poly(A) tail stimulates degradation by the exosome. The TRAMP complex contains the poly(A) polymerase Trf4p, the RNA-binding protein Air2p, and the DExH RNA helicase Mtr4p. The role of Mtr4p in RNA degradation processes involving the TRAMP complex has been unclear. Here we show through a genetic analysis that MTR4 is required for degradation but not for polyadenylation of hypomodified tRNA(iMet). A suppressor of the trm6-504 mutation in the tRNA m(1)A58 methyltransferase (Trm6p/Trm61p), which causes a reduced level of tRNA(iMet), was mapped to MTR4. This mtr4-20 mutation changed a single amino acid in the conserved helicase motif VI of Mtr4p. The mutation stabilizes hypomodified tRNA(iMet) in vivo but has no effect on TRAMP complex stability or polyadenylation activity in vivo or in vitro. We further show that purified recombinant Mtr4p displays RNA-dependent ATPase activity and unwinds RNA duplexes with a 3'-to-5' polarity in an ATP-dependent fashion. Unwinding and RNA-stimulated ATPase activities are strongly reduced in the recombinant mutant Mtr4-20p, suggesting that these activities of Mtr4p are critical for degradation of polyadenylated hypomodified tRNA(iMet).

Figures

FIGURE 1.
FIGURE 1.
A low-copy plasmid bearing MTR4 complements the mutant phenotypes of a trm6-504/sup3/gcn2-101 strain. (A) Wild-type (Y200), trm6-504 (Y190), and trm6-504/sup3 (Sup3) strains were transformed with YCpLac33 or a low-copy number MTR4 plasmid. Strains were grown as patches on synthetic complete (SC) plates lacking uracil at 30°C and replica-plated to SC plates lacking uracil and histidine supplemented with 30 mM 3-aminotriazole (3-AT) or SC plates lacking uracil and incubated 30°C and 37°C, respectively. (B) Northern blot analysis of total RNA (10 μg) isolated from the same strains described in A grown at 30°C in SC medium lacking uracil. Hybridization with probes JA11 (tRNAi Met) and JA151 (tRNACCA Leu) were performed as described in Materials and Methods, followed by autoradiography or PhosphorImager analysis and quantification using Image Quant software. tRNAi Met was normalized to the amount of tRNACCA Leu in the same sample and is expressed as percentage of the wild type.
FIGURE 2.
FIGURE 2.
Schematic representation of MTR4. (A) (Light gray box) The MTR4 ORF; (black and dark gray boxes) the conserved motifs found in most RNA helicases. The conserved amino acids in motif II and motif VI are shown under the boxes. The amino acid mutated in mtr4-20 is indicated in motif VI (methionine to isoleucine). (B) Alignment of Motif VI between members of the DExH subfamily in yeast. The shaded background designates identical amino acids in motif VI. The methionine (bold), which is mutated in mtr4-20p to isoleucine and highly conserved among members of the Ski2p subfamily, is indicated in bold.
FIGURE 3.
FIGURE 3.
Pre-tRNAi Met lacking m1A58 is polyadenylated in trm6-504/ mtr4-20. (Lanes 4–6) Total RNA (5.0 μg) or (lanes 1–3) poly(A)+ RNA (2.0 μg) isolated from the indicated strains cultured at 30°C was separated by denaturing polyacrylamide gel electrophoresis and transferred to a membrane. Hybridization was performed with radiolabeled probe JA11 (tRNAi Met), and tRNAi Met was visualized by autoradiography.
FIGURE 4.
FIGURE 4.
Mtr4-20p and Trf4p are in a complex. Tap-tagged Mtr4p (Y358) and Mtr4-20p (Y359) were purified from whole cell extracts (Materials and Methods). Equivalent amounts of affinity-purified Mtr4p, as determined by immunoblot using anti-Mtr4p polyclonal serum, and whole cell extracts (40 μg) were separated by SDS-PAGE and transferred to a membrane for immunoblot. Rabbit anti-Trf4p or anti-Mtr4p polyclonal serums were used to probe for the respective proteins followed by hrp-coupled goat anti-rabbit IgG antibodies and ECL for detection. Mtr4p with altered mobility due to the presence of the Tap-tag is indicated with an asterisk. (Lane 1) trf4Δ (F22), (lanes 2,3) purified Mtr4p complexes from Tap-Mtr4 (Y358) and Tap-mtr4-20, and (lane 4) the equivalent amount of protein found in lanes 2 and 3 except purified using extract containing untagged Mtr4p (Y200).
FIGURE 5.
FIGURE 5.
Mutant and wild-type Mtr4p complexes possess robust polyadenylation activity in vitro. Polyadenylation of radiolabeled synthetic initiator tRNA was conducted with purified complexes containing wild-type Mtr4p (MTR4), Mtr4–20p (mtr4-20), or proteins purified from an extract containing no TAP-tag (Mock). Reactions were incubated from 0 to 90 min with (1 mM) or without ATP at 30°C and once stopped by addition of formamide loading dye separated by denaturing polyacrylamide electrophoresis, dried, and detected by autoradiography. (Lane 1) A control reaction with no protein added.
FIGURE 6.
FIGURE 6.
Purification of recombinant wild-type and mutant Mtr4p. 6xHis-tagged Mtr4p was expressed in E. coli and purified using TALON metal affinity resin and anion exchange chromatography (Materials and Methods). Equal amounts of purified protein, as determined by Bradford assay, were resolved on 4%–12% gradient SDS-PAGE gel. (A) Silver staining and (B) Western blot with anti-His antibody were used to visualize and quantify the amount of purified protein in each preparation. (Lanes 1,4) WT Mtr4p; (lanes 2,5) Mtr4-20p; (lanes 3,6) Mtr4-21p contains the changes DEvH to AAvH.
FIGURE 7.
FIGURE 7.
Mtr4p but not Mtr4-20p and Mtr4-21p unwinds RNA duplexes with a 3′-to-5′ polarity in an ATP-dependent fashion. (A) Representative PAGE of an unwinding reaction of a 16-bp duplex [S16-3′] with a 3′-single-stranded overhang by wild-type Mtr4p. Cartoons on the left represent the mobility of duplex and the single-stranded radiolabeled RNA; the asterisk indicates the radiolabel. Reactions were assembled with Mtr4p and without ATP, and an aliquot removed at time zero. After ATP addition, aliquots were removed at the times shown. Complete dissociation of the duplex is shown after incubating the RNA briefly at 95°C. (B) Time courses of unwinding reactions by wild-type Mtr4p for 16-bp duplexes with 3′- and 5′-single-stranded overhangs with and without ATP (inset). Sequences of duplexes and single stranded overhangs were identical (Materials and Methods). Significant unwinding was seen only for the substrate with the 3′-overhang (•). The solid line through these points indicates the best fit to a first-order reaction (Yang and Jankowsky 2005), yielding the reaction amplitude A = 0.65 ± 0.02, and the observed unwinding rate constant kunw = 0.057 ± 0.003 min−1. (C) Representative PAGE of unwinding reactions with a 19-bp substrate with a 3′-overhang [S19-3′] with wild-type Mtr4p and Mtr4-20p and Mtr4-21p. For the mutant proteins, only aliquots removed after 60 min are shown. Wild-type Mtr4p unwinds the 19-bp substrate with an observed rate constant (kunw = 0.054 ± 0.007 min−1) highly similar to that seen with the 16-bp duplex, but the reaction amplitude is lower (A = 0.39 ± 0.03) than for the 16-bp duplex.
FIGURE 8.
FIGURE 8.
Mtr4-20p is defective in tRNA-dependent ATPase activity. Purified recombinant Mtr4p was incubated with [α-32P]ATP in the presence of 2.5 μg, 5 μg, or absence of E. coli total tRNA. Aliquots of reactions were collected and stopped by the addition of 0.1% SDS, 5 mM EDTA after 15 min and 60 min at 30°C. Visualization of ATP, ADP, AMP, and Pi was accomplished by incubating 1.0 μCi of [α-32P]ATP and 0.025 U of CIAP together for 15 min at 37°C. Samples were resolved by thin layer chromatography (Materials and Methods), and reaction products were visualized by autoradiography. The positions of ATP, ADP, AMP, and Pi are indicated.

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