LRPPRC and SLIRP interact in a ribonucleoprotein complex that regulates posttranscriptional gene expression in mitochondria

Abstract

Mutations in LRPPRC are responsible for the French Canadian variant of Leigh syndrome (LSFC), a neurodegenerative disorder caused by a tissue-specific deficiency in cytochrome c oxidase (COX). To investigate the pathogenic mechanism of disease, we studied LRPPRC function in LSFC and control fibroblasts. The level of mutated LRPPRC is reduced in LSFC cells, and this results in decreased steady-state levels of most mitochondrial mRNAs, but not rRNAs or tRNAs, a phenotype that can be reproduced by siRNA-mediated knockdown of LRPPRC in control cells. Processing of the primary transcripts appears normal. The resultant defect in mitochondrial protein synthesis in LSFC cells disproportionately affects the COX subunits, leading to an isolated COX assembly defect. Further knockdown of LRPPRC produces a generalized assembly defect in all oxidative phosphorylation complexes containing mtDNA-encoded subunits, due to a severe decrease in all mitochondrial mRNAs. LRPPRC exists in a high-molecular-weight complex, and it coimmunoprecipitates with SLIRP, a stem-loop RNA-binding protein. Although this interaction does not depend on mitochondrial mRNA, both proteins show reduced stability in its absence. These results implicate LRPPRC in posttranscriptional mitochondrial gene expression as part of a ribonucleoprotein complex that regulates the stability and handling of mature mRNAs.

Figure 1.

The COX assembly defect in LSFC fibroblasts is accompanied by a decreased synthesis of several mitochondria-encoded polypeptides. Control and LSFC fibroblasts were analyzed by BN-PAGE (A) and by Western blotting (B). In A, each of the five OXPHOS complexes (Co I–V) was visualized with a subunit-specific antibody. In B, the blots were incubated with antibodies against the proteins indicated at the right of the panel. The 70-kDa subunit of complex II was used as a loading control. (C) Control and LSFC fibroblasts were pulse-labeled with [³⁵S]methionine and cysteine in the presence of anisomycin, an inhibitor of cytoplasmic protein synthesis, and subsequently were chased for 10 min (PULSE) or overnight (CHASE). Fifty micrograms of total protein were run on a 15–20% polyacrylamide gradient gel. The seven subunits of complex I (ND), one subunit of complex III (cyt b), three subunits of complex IV (COX), and two subunits of complex V (ATP) are indicated at the left of the figure. (D) Quantification of the synthesis of individual mitochondria-encoded polypeptides in fibroblasts from eight LSFC patients (four fibroblast lines in triplicate; four lines as a single experiment).

Figure 2.

Localization of mutant LRPPRC to mitochondria in LSFC fibroblasts. Fibroblasts from control and one LSFC patient were grown on coverslips and incubated with antibodies against LRPPRC and against cytochrome c followed by incubation with secondary antibodies coupled to red and green fluorescent dyes, respectively. The overlay of these two images demonstrates the mitochondrial localization of mutant LRPPRC in patient fibroblasts and confirms the reduction in mutant LRPPRC levels in LSFC fibroblasts.

Figure 3.

Reduction in the steady-state levels of mitochondrial mRNAs in LSFC fibroblasts. (A) Northern blot analysis was carried out with total RNA extracted from control and LSFC fibroblasts. Hybridization was performed with probes specific for the mitochondrial mRNAs encoding the three COX subunits, four of the complex I subunits (ND), the bicistronic mRNA encoding the complex V subunits (ATP6/8) and, as a loading control, with a probe for the 12S mitochondrial rRNA. (B) Quantification of mitochondrial mRNA levels in fibroblasts from four LSFC patients normalized to 12S RNA levels (3–8 replicates per mRNA).

Figure 4.

Decreased assembly of the OXPHOS complexes in control and LSFC fibroblasts after knockdown of LRPPRC. Control and LSFC fibroblasts were transiently transfected with one of two different siRNA constructs specific to LRPPRC (S1173 and S3017) or with a fluorescent control siRNA (Alexa) and analyzed by BN-PAGE (A) and SDS-PAGE (B).

Figure 5.

Generalized decrease in mitochondrial translation and in the levels of mitochondrial mRNAs in control and LSFC fibroblasts after LRPPRC knockdown. Fibroblasts from one control and one LSFC patient were transiently transfected either with one of two different siRNA constructs specific to LRPPRC (S1173 and S3017) or with a fluorescent control siRNA (Alexa). On day 6, transfected and untransfected cells were either pulse-labeled with [³⁵S]methionine and cysteine (A) or total RNA was extracted and analyzed by Northern blot (B). (A) The seven subunits of complex I (ND), one subunit of complex III (cyt b), three subunits of complex IV (COX), and two subunits of complex V (ATP) are indicated at the left of the figure. (B) Hybridization was performed with probes specific for the mitochondrial mRNAs encoding the three COX subunits, the ND1 subunit of complex I, and the bicistronic mRNA encoding the complex V subunits (ATP6/8) and with probes for the 12S and 16S mitochondrial rRNAs.

Figure 6.

Normal levels of mitochondrial tRNAs in fibroblasts from LSFC patients and in fibroblasts in which LRPPRC was knocked down. Total RNA was extracted from control cells transiently transfected either with one of two different siRNA constructs specific to LRPPRC (S1173 and S3017) or with a fluorescent control siRNA (Alexa) and from untransfected controls and LSFC patients, after which 5 μg total RNA/sample were run on a 10% polyacrylamide gel containing 7 M urea. After transfer to membrane, hybridization was performed with oligonucleotide probes complementary to the mitochondrial tRNAs for Lys, Glu, Gln, Trp, and Val and, as a loading control, the cytoplasmic tRNA for Glu (cyt Glu), as indicated at the right of the figure.

Figure 7.

LRPPRC exists in a higher molecular weight complex and interacts with SLIRP. (A) Mitochondria from control fibroblasts were separated by size exclusion and the individual fractions were analyzed by SDS-PAGE. The molecular weights of the individual fractions were calculated from the elution profile of a set of standards. (B) Coimmunoprecipitation of LRPPRC and SLIRP. Mitochondria from control fibroblasts were extracted with 1% sodium deoxycholate, and the extract (preclear fraction) was incubated overnight with naked beads to reduce nonspecific binding to the beads during the subsequent IP reaction. The cleared extract (input fraction) was divided among naked beads (control IP), beads cross-linked with anti-LRPPRC antibody (LRPPRC IP), and beads cross-linked with anti-SLIRP antibody (SLIRP IP). “Unbound” refers to the fractions collected after the IP reactions were allowed to proceed overnight. Subsequently, the beads were washed (wash fractions) and eluted with acidic glycine (eluate fractions). Individual fractions were then analyzed by immunoblotting with antibodies against LRPPRC and SLIRP. (C) The LRPPRC–SLIRP complex contains mitochondrial mRNAs. Immunoprecipitation from control mitochondrial extracts was performed with the antibody against LRPPRC as described in B, and RNA was then extracted from the immunoprecipitate (pellet) and unbound (supernatant) fractions and was used to amplify COX I and COX II mRNA sequences by RT-PCR. Mitochondrial mRNA, but not mtDNA, was specifically found in the LRPPRC immunoprecipitate. (D) LRPPRC and SLIRP are detected as part of the same high-molecular-weight complex on 2D-PAGE. Extracts of control and LSFC fibroblasts were run on a nondenaturing gel (BN-PAGE) after which the individual lanes were excised and run on a second, denaturing gel (SDS-PAGE). Antibodies against the subunits of all five OXPHOS complexes, LRPPRC, and SLIRP were used, as indicated at the right of the gel. In the upper middle panel analysis of three subunits of COX shows reduced levels in LSFC cells. The lower middle panel was probed for the indicated complexes after stripping. The COX I signal is the residual signal from the upper middle blot. The sizes of the LRPPRC–SLIRP-containing complexes are indicated at the top of the figure.

Figure 8.

LRPPRC and SLIRP are interdependent and show reduced stability in the absence of mitochondrial mRNA. (A and B) Western blot analyses of SLIRP and LRPPRC in patients with reduced endogenous levels of LRPPRC (A) and human 143B rho⁰ cells (whole cells or mitochondrial fractions as indicated; B) show coordinate decreases in the levels of SLIRP and LRPPRC. Antibodies against the 70-kDa subunit of complex II and against porin were used as loading controls. The lanes labeled COX patients represent patients with uncharacterized COX deficiencies. (C) Coimmunoprecipitation of LRPPRC and SLIRP from mitochondrial extracts of rho⁰ cells. Mitochondria from human rho⁰ cells were extracted and used for IP reactions with the anti-SLIRP antibody as described in Figure 7B. The individual fractions identified at the top of the gel were analyzed by immunoblotting with the anti-LRPPRC and anti-SLIRP antibodies.