Rpm2p: separate domains promote tRNA and Rpm1r maturation in Saccharomyces cerevisiae mitochondria

Abstract

Rpm2p is a protein subunit of yeast mitochondrial RNase P and is also required for the maturation of Rpm1r, the mitochondrially-encoded RNA subunit of the enzyme. Previous work demonstrated that an insertional disruption of RPM2, which produces the C-terminally truncated protein Rpm2-DeltaCp, supports growth on glucose but cells lose some or all of their mitochondrial genome and become petite. These petites, even if they retain the RPM1 locus, lose their ability to process the 5'-ends of mitochondrial tRNA. We report here that if strains containing the truncated RPM2 allele are created and maintained on respiratory carbon sources they have wild-type mitochondrial genomes, and a significant portion of tRNA transcripts are processed. In contrast, precursor Rpm1r transcripts accumulate and mature Rpm1r is not made. These data show that one function of the deleted C-terminal region is in the maturation of Rpm1r, and that this region and mature Rpm1r are not absolutely required for RNase P activity. Finally, we demonstrate that full activity can be restored if the N-terminal and C-terminal domains of Rpm2p are supplied in trans.

Figure 1

The rpm2-734::LEU2 allele supports respiratory growth if spores are grown directly on medium with glycerol/ethanol. (A) A schematic representation of the diploid RPM2/rpm2-734::LEU2. Numbers indicate amino acid residues in Rpm2p. (B) Diploid cells were sporulated and tetrads were dissected on YPGE plates. The small colony size segregated with the LEU2 allele. (C) Mitochondrial proteins were isolated from wild-type and rpm2-734::LEU2 cells, separated by Tris–acetate–polyacrylamide gel electrophoresis, and analyzed by immunoblotting using anti-Rpm2p polyclonal antibodies. The nuclear-encoded mitochondrial protein Mdh1p was used as a loading control. Lane 1, RPM2; lane 2, rpm2-734::LEU2. Protein molecular mass standards are indicated on the right.

Figure 2

Processing of mitochondrial tRNAs in wild-type and rpm2-734::LEU2 cells. Mitochondrial RNA was isolated from cells grown in glycerol/ethanol medium (YPGE). A northern blot was performed with probes to (A) tRNA_f^Met and tRNA^Phe (top and bottom, respectively), (B) tRNA^Glu and (C) tRNA^Pro. Wild-type (lane 1) and rpm2-734::LEU2 (lane 2). Pre-tRNA and tRNA mark the positions of precursor and mature tRNA, respectively.

Figure 3

rpm2-734::LEU2 cells do not make mature Rpm1r. (A) Rpm1r is transcribed either with tRNA_f^Met and tRNA^Pro from the FP promoter, or with only tRNA^Pro from the SP promoter (top). Vertical arrows correspond to processing events and putative processing intermediates are represented below. The dotted line corresponds to 5′-ends, which can be generated either by cleavage at the 3′-end of tRNA_f^Met or by transcription initiation at SP promoter. Mitochondrial RNA was isolated from cells grown in YPGE. A northern blot was performed with probes to Rpm1r (B) and tRNA^Pro (C). Lane 1, wild-type; lane 2, rpm2-734::LEU2.

Figure 4

Mapping the 5′- and 3′-ends of RPM1 transcripts. (A) 5′-ends were determined by extending a radiolabeled primer complementary to a sequence in Rpm1r. A dideoxy sequencing ladder (lanes 1–4) and extension products from wild-type (lanes 6 and 7, mitochondrial RNA isolated from YPD and YPGE grown cells, respectively), and rpm2-734::LEU2 (lane 8, mitochondrial RNA isolated from YPGE grown cells) RNAs were separated on a 6% denaturing gel. 5′ end represents the mature 5′-end of Rpm1r. SP and FP represent 5′-ends of primary transcripts, which originate at consensus promoter sequences downstream and upstream of the tRNA_f^Met gene, respectively. 3′ Met_f represents the 5′-end of a product that is likely formed by an endonuclease cleavage at the 3′-end of tRNA _f^Met. (B) A single-stranded DNA probe was hybridized with mitochondrial RNA isolated from YPGE grown cells. The samples were digested with S1 nuclease, and protected fragments were separated on a 6% polyacrylamide denaturing gel. Probe alone (lanes 1 and 2, in the absence or presence of S1 nuclease, respectively); probe plus S1 nuclease in the presence of wild-type RNA (lane 3) and increasing amounts of rpm2-734::LEU RNA (10, 20 and 30 µg in lanes 4, 5 and 6, respectively). 3′ end represents the mature 3′end of Rpm1r. Intermediate marks a putative processing intermediate discussed in the text.

Figure 5

N- and C-terminal truncated Rpm2p proteins function in trans. (A) A schematic representation of Rpm2 proteins. Numbers indicate amino acid residues. The filled box represents a putative mitochondrial targeting sequence. (B) RPM2, ΔC + ΔN or ΔC(–20) + ΔN alleles on different low-copy plasmids were introduced into a Δrpm2 haploid strain containing RPM2 on a URA3 plasmid and serial dilutions of transformants plated onto 5-FOA plates to assess growth in the absence of wild-type RPM2. (C) Strains were grown overnight in liquid YPGE medium and serial dilutions plated on YPD and YPGE. Cells were incubated 2 and 4 days for YPD and YPGE plates, respectively. (D) Mitochondrial proteins were isolated from the glycerol/ethanol grown cells shown in (C), separated on Tris–acetate–polyacrylamide gel electrophoresis and analyzed by immunoblotting using anti-Rpm2p polyclonal antibodies. The nuclear-encoded mitochondrial protein Mdh1p was used as a loading control. Lane 1, RPM2; lane 2, ΔC + ΔN; lane 3, ΔC (rpm2-734::LEU2). Protein molecular mass standards are indicated on the right.

Figure 5

N- and C-terminal truncated Rpm2p proteins function in trans. (A) A schematic representation of Rpm2 proteins. Numbers indicate amino acid residues. The filled box represents a putative mitochondrial targeting sequence. (B) RPM2, ΔC + ΔN or ΔC(–20) + ΔN alleles on different low-copy plasmids were introduced into a Δrpm2 haploid strain containing RPM2 on a URA3 plasmid and serial dilutions of transformants plated onto 5-FOA plates to assess growth in the absence of wild-type RPM2. (C) Strains were grown overnight in liquid YPGE medium and serial dilutions plated on YPD and YPGE. Cells were incubated 2 and 4 days for YPD and YPGE plates, respectively. (D) Mitochondrial proteins were isolated from the glycerol/ethanol grown cells shown in (C), separated on Tris–acetate–polyacrylamide gel electrophoresis and analyzed by immunoblotting using anti-Rpm2p polyclonal antibodies. The nuclear-encoded mitochondrial protein Mdh1p was used as a loading control. Lane 1, RPM2; lane 2, ΔC + ΔN; lane 3, ΔC (rpm2-734::LEU2). Protein molecular mass standards are indicated on the right.

Figure 6

The C-terminal domain together with the N-terminal domain restores full activity of Rpm2p. Northern analysis of wild-type (lanes 1 and 3), ΔC + ΔN (lanes 2 and 4) and rpm2-734::LEU2 (lane 5) total RNAs. Upper panel shows the membrane probed with a riboprobe to Rpm1r; lower panel shows the membrane probed with a riboprobe to tRNA_f^Met.