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MRM2 encodes a novel yeast mitochondrial 21S rRNA methyltransferase - PubMed

  • ️Tue Jan 01 2002

Comparative Study

MRM2 encodes a novel yeast mitochondrial 21S rRNA methyltransferase

Lionel Pintard et al. EMBO J. 2002.

Abstract

Mitochondria of the yeast Saccharomyces cerevisiae assemble their ribosomes from ribosomal proteins, encoded by the nuclear genome (with one exception), and rRNAs of 15S and 21S, encoded by the mitochondrial genome. Unlike cytoplasmic rRNA, which is highly modified, mitochondrial rRNA contains only three modified nucleotides: a pseudouridine (Psi(2918)) and two 2'-O-methylated riboses (Gm(2270) and Um(2791)) located at the peptidyl transferase centre of 21S rRNA. We demonstrate here that the yeast nuclear genome encodes a mitochondrial protein, named Mrm2, which is required for methylating U(2791) of 21S rRNA, both in vivo and in vitro. Deletion of the MRM2 gene causes thermosensitive respiration and leads to rapid loss of mitochondrial DNA. We propose that Mrm2p belongs to a new class of three eukaryotic RNA-modifying enzymes and is the orthologue of FtsJ/RrmJ, which methylates a nucleotide of the peptidyl transferase centre of Escherichia coli 23S rRNA that is homologous to U(2791) of 21S rRNA. Our data suggest that this universally conserved modified nucleotide plays an important function in vivo, possibly by inducing conformational rearrangement of the peptidyl transferase centre.

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Figures

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Fig. 1. Sequence alignment of Mrm2p from Saccharomyces cerevisiae with potential homologues from different species. Ten proteins have been aligned using ClustalW (Thompson et al., 1994). S.c, Mrm2p from S.cerevisiae (P53123); S.k, Saccharomyces kluyveri; K.m, Kluyveromyces marxianus; K.l, Kluyveromyces lactis; S.b Saccharomyces bayanus; P.a, Pichia angusta; P.f, Plasmodium falciparum (unfinished sequence from the P.falciparum Genome Project: PlasmoDB-http://www.plasmodb.org); C.a, Candida albicans (unfinished sequence from the Stanford Genome Technology Centre–SGTC: http://sequence-www.stanford.edu); S.p, S.pombe (P78860); E.c, FtsJ/RrmJ from E.coli (P28692). All the hemiascomycetous yeast sequences are unfinished sequences from Genolevures Project (http://cbi.labri.u-bordeaux.fr/Genolevures). Identical amino acids are on a black background and chemically equivalent groups are on a grey background. The nine motifs that form the putative AdoMet-binding domain are boxed and noted X and I–VIII. The four residues that are proposed to form the catalytic centre are indicated by a star. Segments predicted to adopt a secondary structure are indicated below the alignment: white tubes, α-helices (α1–5); striped arrows, β-strands (β1–7). The first insertion of 58 residues can also form an α-helix (αi) and a β-strand (βi).

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Fig. 2. Deletion of MRM2 prevents growth on glycerol at 37°C. (A) Wild-type cells (BMA64), mrm2Δ cells (YCB640) and mrm2Δ + p(MRM2) cells (YCB689) were spotted after serial dilutions (1/10 from left to right) on YPD plates or on YP-glycerol plates at 30 or 37°C and incubated for 4 days. (B) Individual cells were grown on YPD plates at 30°C for 4 days, then tested for their ability to grow on YP-glycerol (indicated as a plus or a minus sign). Left panel, mrm2Δ strain (YCB640); right panel, wild-type strain. Lower panel: western blot analysis of the tested strains grown in YPD with an anti-Cox2p antibody. Extracts were prepared from a mrm2Δ strain able to grow on YPGly (lane 1), from a mrm2Δ petite colony (lane 2) and from a wild-type strain (lane 3).

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Fig. 3. Mrm2p is located within mitochondria. (A) Induction of Mrm2p after a shift from glucose- to glycerol-containing medium. Cells expressing Mrm2–protAp (YCB642) were grown in YPD and shifted to glycerol at t = 0. Extracts were prepared at various times thereafter and analysed by western blotting, probed with an anti-mouse antibody to reveal Mrm2–protAp (upper blot), an anti-Cox2p antibody (middle blot) or an anti-Tcm1p antibody (lower blot). (B) Mrm2p is mostly detected in the mitochondrial fraction. Cells (YCB642) were grown in YP-glycerol until stationary phase. Differential centrifugation was performed after breaking the cells to prepare a crude mitochondrial fraction (M) and a post-mitochondrial fraction (PM). Proteins from a total cell extract (T) and from the different fractions were analysed by western blotting with anti-mouse (upper blot), anti-Cox2p (second blot), anti-Pab1p (third blot) and anti-Qsr1p antibodies (lower blot). (C–G) Microscopic examination of cells overexpressing Mrm2–protAp (YCB651) (C, D and F–G) or wild-type untagged cells (E). (C) and (F) show DAPI staining, and (D), (E) and (G) show fluorescein isothiocyanate analysis. (F) and (G) show an enlargement of one cell of (C) and (D).

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Fig. 4. Mrm2p co-sediments with the 21S rRNA. A cellular extract of a strain expressing Mrm2–protAp (YCB642) was fractionated on a sucrose gradient. A continuous A254nm record is presented (top panel). The arrows indicate the peaks of 40S, 60S and 80S subunits. Each fraction was split into two parts: one was TCA precipitated and analysed by western blotting probed with a HRP-conjugated anti-mouse antibody (middle panel) and the other was phenol extracted and analysed by northern blotting probed with o21S1, an oligonucleotide specific for the 21S rRNA (bottom panel).

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Fig. 5. Mrm2p is required for methylating U2791 in vivo. AMV reverse transcriptase was used to extend 32P-labelled primers in the presence of two different concentrations of dNTPs, either 4 µM or 1 mM. Extension products were separated by 12% PAGE containing 7M urea and autoradiographed overnight. Extension was performed on RNA prepared from the indicated strains either with primer o21S1 specific for U2791 (lanes 1–7) or with primer o21S2 specific for G2270 (lanes 8–12). Lanes 1 and 12, unextended primers.

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Fig. 6. Mrm2p methylates U2791 in vitro. Various RNA substrates have been prepared from a mrm2Δ strain (YCB640) and treated (lanes 3–6 and 8–9) or not (lanes 2 and 7) with the affinity-purified Mrm2–protA protein. To test for the MTase activity of Mrm2p, the substrate was either the whole 54S ribosomal subunit (lanes 2–6) or the deproteinized 21S rRNA (lanes 7–11). Then methylation status was determined by primer extension mapping using o21S1 as described in Figure 5. Lane 1, primer alone; lanes 3, 4 and 8 were treated with Mrm2p for 30 min, and lanes 5, 6 and 9 for 60 min; lanes 10 and 11, untreated control RNA from a wild-type strain. To detect the methylated nucleotides, primer extension was performed with 4 µM dNTPs, except lanes 4, 6 and 11, which were incubated with 1 mM dNTPs.

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