C7orf30 specifically associates with the large subunit of the mitochondrial ribosome and is involved in translation

In a comparative genomics study for mitochondrial ribosome-associated proteins, we identified C7orf30, the human homolog of the plant protein iojap. Gene order conservation among bacteria and the observation that iojap orthologs cannot be transferred between bacterial species predict this protein to be associated with the mitochondrial ribosome. Here, we show colocalization of C7orf30 with the large subunit of the mitochondrial ribosome using isokinetic sucrose gradient and 2D Blue Native polyacrylamide gel electrophoresis (BN-PAGE) analysis. We co-purified C7orf30 with proteins of the large subunit, and not with proteins of the small subunit, supporting interaction that is specific to the large mitoribosomal complex. Consistent with this physical association, a mitochondrial translation assay reveals negative effects of C7orf30 siRNA knock-down on mitochondrial gene expression. Based on our data we propose that C7orf30 is involved in ribosomal large subunit function. Sequencing the gene in 35 patients with impaired mitochondrial translation did not reveal disease-causing mutations in C7orf30

Functional consequences of mitochondrial tRNA Trp and tRNA Arg mutations causing combined OXPHOS defects.

Contains fulltext : 83310.pdf (publisher's version ) (Closed access)Combined oxidative phosphorylation (OXPHOS) system deficiencies are a group of mitochondrial disorders that are associated with a range of clinical phenotypes and genetic defects. They occur in approximately 30% of all OXPHOS disorders and around 4% are combined complex I, III and IV deficiencies. In this study we present two mutations in the mitochondrial tRNA(Trp) (MT-TW) and tRNA(Arg) (MT-TR) genes, m.5556G>A and m.10450A>G, respectively, which were detected in two unrelated patients showing combined OXPHOS complex I, III and IV deficiencies and progressive multisystemic diseases. Both mitochondrial tRNA mutations were almost homoplasmic in fibroblasts and muscle tissue of the two patients and not present in controls. Patient fibroblasts showed a general mitochondrial translation defect. The mutations resulted in lowered steady-state levels and altered conformations of the tRNAs. Cybrid cell lines showed similar tRNA defects and impairment of OXPHOS complex assembly as patient fibroblasts. Our results show that these tRNA(Trp) and tRNA(Arg) mutations cause the combined OXPHOS deficiencies in the patients, adding to the still expanding group of pathogenic mitochondrial tRNA mutations.01 maart 20106 p

Proposed model of CIII assembly.

<p>Complex III assembly begins with the translation activation and/or stabilization of cytochrome <i>b</i> (MTCYB) by UQCC1:UQCC2, which then delivers MTCYB to an assembly intermediate containing UQCRQ and UQCRB. This module combines with a module containing CYC1, UQCRH and UQCR10 and a module containing UQCRC2 and UQCRC1. The resulting subcomplex then dimerizes. UQCRFS1 is bound and stabilized by the CIII assembly factor LYRM7, before being incorporated into CIII with the aid of the assembly factor, BCS1L. Finally UQCR11 is added, forming the complete CIII₂. Assembly factors are indicated in gray. Proteins in which mutations are associated with complex III deficiency are bordered in red. The role of TTC19 is yet to be elucidated, although it is likely to be involved in early complex III assembly. Model adapted and updated from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004034#pgen.1004034-FernandezVizarra2" target="_blank">[67]</a>.</p

The c.214-3C>G mutation causes a severe <i>UQCC2</i> splicing defect.

<p>(A) Gel electrophoresis of full-length <i>UQCC2</i> RT-PCR products from fibroblasts grown in the absence of cycloheximide. Two prominent bands are seen in P^UQCC2 whereas only one is observed in the control. (B) Schematic diagram shows the wild-type (WT) mRNA structure and the two splice variants (1 and 2) observed in P^UQCC2. (C) Sequence chromatograms of cloned RT-PCR products show that the upper product in P^UQCC2 retains 108 bases of intronic sequence due to the use of a cryptic acceptor site, and that the lower product in P^UQCC2 lacks 14 bases of exonic sequence due to the use of an alternative cryptic acceptor site. Splice site prediction scores are from Human Splicing Finder v2.4.1 (<a href="http://www.umd.be/HSF/" target="_blank">http://www.umd.be/HSF/</a>). (D) qRT-PCR analysis using an assay that detects the exon 2/3 junction of <i>UQCC2</i> (normalized to the endogenous control <i>HPRT1</i>) demonstrates P^UQCC2 fibroblasts have only 2% wild-type <i>UQCC2</i> expression relative to controls (C¹–C⁴). P^UQCC2(1) and P^UQCC2(2) represent separate fibroblast subcultures.</p

Depletion of the UQCC2 binding partner, UQCC1, affects complex III assembly.

<p>(A) SDS-PAGE and western blot analysis of mitochondrial extracts from HEK293 cells transfected with <i>UQCC1</i> siRNA shows lower levels of complex III subunits UQCRFS1, UQCRC1 and UQCRC2. Subunits of complex I (ND1), complex II (SDHA), complex IV (COX1) and complex V (ATP5α) are not affected by <i>UQCC1</i> knockdown. (B) BN-PAGE of HEK293 cells transfected with UQCC1 siRNA show reduced levels of holocomplex III (UQCRC2) and a mild effect on complex I in gel activity (IGA) and complex I holocomplex levels (NDUFA9). Levels of other OXPHOS complexes, complex II (SDHB), complex IV (COX2) and complex V (ATP5α) are not affected. Mock transfected cells were used as control). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004034#pgen.1004034.s007" target="_blank">Figure S7B</a>-C for the quantification of the immunoreactive bands. (C) 2D BN-PAGE of HEK293 cells depleted of UQCC1 or cyclophilin B with indicated antibodies. The holocomplex III dimer is indicated with a line labeled CIII₂. To the right are lower molecular weight subcomplexes: UQCRC1-containing subcomplex (1) and, likely, monomeric UQCRFS1 (2). Lauryl maltoside was used to solubilize OXPHOS complexes in parts B and C. (D) Respiratory chain enzyme activity measurements of HEK293 cells transfected with <i>UQCC1</i> and <i>cyclophilin B</i> siRNAs. Mock transfected cells were set at 100%. Error bars indicate one standard deviation. Complex I ubiquinone reducing part (CI-Q), complexes II–V (CII-V) and combined activity of complex II and III (SCC) were measured relative to the activity of citrate synthase (CS).</p

<i>UQCC2</i> mutations are responsible for the complex III defect in P^UQCC2.

<p>Fibroblasts from Control, P^UQCC2 with mutations in <i>UQCC2</i> and P^CONTROL with mutations in a complex III subunit gene (and no <i>UQCC2</i> mutation) were transduced with wild-type <i>UQCC2</i> mRNA. (A) Representative SDS-PAGE western blot shows reduced UQCC2 in P^UQCC2 and increased UQCC2 expression following <i>UQCC2</i> transduction. VDAC1 acts as a loading control. UQCRFS1 protein is reduced in both complex III deficient patients and restored in P^UQCC2, but not P^CONTROL, with <i>UQCC2</i> transduction. VDAC1 acts as a loading control. (B) The intensity of immunostained UQCRFS1 relative to VDAC1 before and after transduction with <i>UQCC2</i> was quantified by densitometry. Error bars indicate 1 s.e.m. for 3 independent transductions and the asterisk indicates p<0.05, two way ANOVA.</p

Lack of UQCC2 is associated with aberrant complex III assembly, subunit expression and UQCC1 stability.

<p>(A) BN-PAGE immunoblotting of mitochondria lysed in 1% Triton X-100, using antibodies against the NDUFA9 subunit of complex I, the SDHA subunit of complex II, the UQCRC1 subunit of complex III and the COX1 subunit of complex IV shows reduced complex III assembly in P^UQCC2. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004034#pgen.1004034.s006" target="_blank">Figure S6A</a> for quantification of immunoreactive bands. (B) SDS-PAGE and western blotting of mitochondrial lysates from patient fibroblasts demonstrate a marked deficiency of UQCC2 and UQCC1, a mild deficiency in the UQCRC2 subunit of complex III, and a more pronounced deficiency of the UQCRC1 and UQCRFS1 subunits of complex III. The P^CONTROL cell line with mutations in a complex III subunit gene showed a similar profile of complex III subunit instability but had levels of UQCC2 and UQCC1 comparable to the wild-type control. The complex II subunit SDHB and mitochondrial VDAC1 protein act as loading controls. Vertical bars indicate immunoblots performed using the same membrane. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004034#pgen.1004034.s006" target="_blank">Figure S6A</a> for quantification of immunoreactive bands. (C) Mitochondrial lysates of HEK293 cells transfected with siRNA targeting <i>UQCC2</i> analyzed by SDS-PAGE and western blotting showed reduced levels of UQCC2 and UQCC1 proteins. As control, <i>cyclophilin B</i> knockdown and mock transfected cells were used. The asterisk indicates a non-specific, cross-reactive species. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004034#pgen.1004034.s007" target="_blank">Figure S7A</a> for quantification of immunoreactive bands.</p

UQCC2 interacts with mitochondrial protein UQCC1.

<p>(A) SDS-PAGE analysis of HEK293 cellular fractions shows that UQCC1 is enriched in the mitochondrial fraction, similar to the mitochondrial protein TOM20. A cytosolic marker creatine kinase B-type (CK-B) was used. TC: Total Cell, Cyt: Cytoplasmic fraction, Mit: Mitochondrial fraction. (B) Proteinase K protection assay performed using mitochondria with digitonin-permeabilized outer membranes shows localization of UQCC1 within the mitochondrial inner membrane. UQCC1, unlike outer membrane localized TOM20 and the inter-membrane localized part of OXA1L, is protected from proteolysis and degraded only after the inner membrane is dissolved with Triton X-100. Western blot analysis of single step affinity purified (C) UQCC2- and (D) UQCC1-TAP from doxycycline-induced HEK293 cells shows that UQCC1 co-purifies with UQCC2-TAP and UQCC2 co-purifies with UQCC1-TAP. Additional probing of the membranes for the complex III structural subunits UQCRC1, UQCRC2, UQCRFS1 and mitochondrial ribosomal subunits MRPS22 and MRPL12 did not reveal co-elution of these proteins. Asterisks with these subunits, including the one with UQCRFS1, correspond to bands at different heights that result from previous incubations. Complex II subunit SDHA was used to rule out non-specific protein binding. Non-induced cells were used as control. Antibodies used are indicated at the left. Arrowheads indicate endogenous UQCC1 and UQCC2.</p