The DEAD box protein Mrh4 functions in the assembly of the mitochondrial large ribosomal subunit

Abstract

Proteins in a cell are universally synthesized by ribosomes. Mitochondria contain their own ribosomes, which specialize in the synthesis of a handful of proteins required for oxidative phosphorylation. The pathway of mitoribosomal biogenesis and factors involved are poorly characterized. An example is the DEAD box proteins, widely known to participate in the biogenesis of bacterial and cytoplasmic eukaryotic ribosomes as either RNA helicases or RNA chaperones, whose mitochondrial counterparts remain completely unknown. Here, we have identified the Saccharomyces cerevisiae mitochondrial DEAD box protein Mrh4 as essential for large mitoribosome subunit biogenesis. Mrh4 interacts with the 21S rRNA, mitoribosome subassemblies, and fully assembled mitoribosomes. In the absence of Mrh4, the 21S rRNA is matured and forms part of a large on-pathway assembly intermediate missing proteins Mrpl16 and Mrpl39. We conclude that Mrh4 plays an essential role during the late stages of mitoribosome assembly by promoting remodeling of the 21S rRNA-protein interactions.

Figure 1. Mrh4 is essential for OXPHOS system assembly and function. See also Table S1

(A) Growth test using serial dilutions of the indicated strains in complete media containing fermentable (YPD) or non-fermentable (YPEG) carbon sources. The plates were incubated at 30°C and the pictures taken after 2 days of growth. (B) Endogenous cell respiration measured polarographically and cytochrome c oxidase (COX), NADH cytochrome c reductase (NCCR), ATP synthase (ATPase) activities measured spectrophotometrically in the indicated strains. Error bars represent the mean ± SD. (C) Total mitochondrial cytochrome spectra. Mitochondria from WT and Δmrh4 strains were extracted with potassium deoxycholate under conditions that quantitatively solubilize all of the cytochromes (36). Difference spectra of the reduced (sodium dithionite) versus oxidized (potassium ferricyanide) extracts were recorded at room temperature. The absorption bands corresponding to cytochromes a and a₃ have maxima at 603 nm (a and a₃); the maxima for cytochrome b (b) and for cytochrome c and c₁ (c and c₁) are 560 and 550 nm, respectively. (D) Immunoblot analyses of the steady-state levels of Mrh4 and the indicated OXPHOS subunits in WT and Δmrh4 strains expressing the mtDNA instability suppressor (SUP). An antibody against Porin was used as a loading control.

Figure 2. Mrh4 is required for mitochondrial protein synthesis and not for mRNA and rRNA processing or stability. See also Fig. S1

(A) In vivo mitochondrial protein synthesis in the indicated wild-type (WT) and Δmrh4 strains, expressing either the mtDNA instability suppressor (SUP), ectopic VAR1 or both. The strains were grown at 30°C and pulsed for 5 or 10 min at the same temperature with [³⁵S]methionine in the presence of cycloheximide to inhibit cytoplasmic protein synthesis. (B and C) Northern blot analyses of total RNA probed for (B) COX1, COB and (C) 15S and 21S rRNA. After processing, the membranes were exposed to X-ray film. The lower panels show the densitometry values obtained by using the histogram function of the Adobe Photoshop program on digitalized images. The values were normalized by the signal of ACT1 as the loading control and expressed relative to the control.

Figure 3. Characterization of temperature sensitive (ts) mrh4 mutants indicates a role for Mrh4 in assembly of the mitochondrial translational apparatus. See also Fig. S2

(A) Scheme representing the two domains and the conserved motifs present in Mrh4, typical of DEAD box helicases (Parsyan et al., 2011). (B) Alignment showing the ATP binding motifs in Prp5 and Mrh4. The amino acids mutated in each case to yield temperature sensitive (ts) proteins are labeled in red. (C) Growth test using serial dilutions of the indicated strains in complete media containing fermentable (glucose, YPD) or non-fermentable (ethanol-glycerol, YPEG) carbon sources. The plates were incubated at the permissive 24°C or non-permissive 38°C and the pictures taken after 2 days of growth. (D) In vivo mitochondrial protein synthesis in the indicated wild-type (WT) and ts2 strains. The strains were grown at the permissive (24°C) or non-permissive (38°C) temperatures and aliquots pulsed for 15 min at each temperature with [³⁵S]methionine in the presence of cycloheximide to inhibit cytoplasmic protein synthesis.

Figure 4. Mrh4 is a mitochondrial matrix protein peripherally bound to the inner membrane interacting with the 54S large mitoribosomal subunit. See also Fig. S3 and Table S2

(A) Isolated WT mitochondria were fractionated into soluble (S) and membrane-bound (P) mitochondrial proteins by brief sonication and centrifugation. The pellet was submitted to alkaline extraction to allow the separation of the extrinsic proteins present in the supernatant (Cs) from the intrinsic proteins in the pellet (Cp). Equivalent volumes of each fraction were analyzed by immunoblotting using antibodies against Mrh4, the intermembrane space soluble protein Cyt b₂, the inner membrane extrinsic protein Mss51 and the inner membrane intrinsic protein Cox3. The right panel represents a proteinase K protection assay in mitochondria (Mt) and mitoplasts (Mp) prepared by hypotonic swelling of mitochondria. The samples were analyzed by immunoblotting using antibodies against Mrh4, Cyt b₂ and Cox4 (protein facing the matrix). (B) Sucrose gradient sedimentation analyses of Mrh4 and ribosomal subunits on mitochondrial extracts prepared from the WT and Δmrh4 strains in the presence of 1% digitonin and the conditions stated. (C) Immunoblot analyses of the steady-state levels of Mrh4 and the indicated r-proteins in WT mitochondria isolated from untreated cells (U) or cells grown in the presence of acriflavin (AF) or ethidium bromide (EtBr) for the indicated times. An antibody against Porin was used as a loading control. (D) Sucrose gradient sedimentation analyses of Mrh4 and ribosomal subunits on mitochondrial extracts prepared from the strains in panel (C) in the presence of 1% digitonin and the conditions stated. (E) Immunoprecipitation of Mrh4 from WT extracts of chemically cross-linked mitochondria as explained in the experimental procedures section. (F) PCR analyses of reserve transcribed Mrh4-co-immunoprecipitated RNA from formaldehyde- or UV-induced cross-linked or non-cross-linked mitochondria.

Figure 5. The helicase activity of Mrh4 is required for function in vivo

(A) Scheme showing the ATP binding and DEAD-Box motifs in Mrh4 and the mutated amino acids. (B) Growth test using serial dilutions of the indicated strains in complete media containing fermentable (YPD) or non-fermentable (YPEG) carbon sources. The plates were incubated at 30°C and the pictures taken after 2–3 days of growth. ep, empty plasmid. (C) In vivo mitochondrial protein synthesis (MPS) in wild-type (WT) and Δmrh4 strains expressing the indicated MRH4 alleles. The bottom panel shows immunoblot analysis of Mrh4 levels using Porin as a loading control.

Figure 6. Mrh4 is essential for a late-stage step in 54S mitoribosome assembly. See also Fig. S4 and Tables S2–S5

(A) Sucrose gradient sedimentation profiles of Mrp10 from the small ribosomal subunit (SSU) and Mrpl40 from the large ribosomal subunit (LSU) in mitochondrial extracts prepared in the presence of 1% digitonin and the indicated conditions from the WT and Δmrh4 strains expressing Sup and ectopic VAR1. (B) Steady-state levels of LSU proteins and assembly factors in the indicated strains analyzed by immunoblotting. (C and D) Sucrose gradient sedimentation analyses of SSU and LSU proteins and LSU assembly factors (AF) in mitochondrial extracts from the indicated strains prepared in the presence of 1% digitonin, 25 mM KCl and either (C) 0,5 mM Mg²⁺ or (D) 5 mM EDTA. The fractions were used to measure total RNA concentration (top) and to analyze the distribution of Mrh4 and the ribosomal proteins by immunoblotting (bottom). (E) Mitoribosomes from WT and Δmrh4 strains purified in the presence of 10 mM Mg²⁺ and 500 mM NH₄Cl were fractionated on a sucrose gradient using a BRB-188 density gradient fractionator (Brandel, Gaithersburg, MD). For each strain, a continuous A254nm record is presented (top). The dashed line indicates the probable position of the 21S rRNA peak based in the immunoblotting results presented at the bottom.

Figure 7. The Δmrh4 pre-54S particle is an on-pathway intermediate

Sucrose gradient sedimentation analyses of [³H]UTP-labeled rRNA in (A) WT and Δmrh4 mitochondrial extracts and Δmrh4 extracts incubated with ρ⁰ extracts containing Mrh4 and r-proteins; (B) mitochondria purified from the mrh4-ts2 mutant strain grown either at 24°C or at 38°C and aliquots of 38°C-grown mitochondria incubated at 24°C for 1h or 2h. The graph represents the [³H] count in each fraction. In the lower panels, the fractions in each case were used for immunoblot analysis of Mrh4 and the indicated r-proteins.