Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome  Nicole J. Lake, Bryn D. Webb, David A. Stroud, Tara R. Richman, Benedetta Ruzzenente, Alison G. Compton, Hayley S. Mountford, Juliette Pulman, Coralie Zangarelli, Marlene Rio, Nathalie Boddaert, Zahra Assouline, Mingma D. Sherpa, Eric E. Schadt, Sander M. Houten, James Byrnes, Elizabeth M. McCormick, Zarazuela Zolkipli-Cunningham, Katrina Haude, Zhancheng Zhang, Kyle Retterer, Renkui Bai, Sarah E. Calvo, Vamsi K. Mootha, John Christodoulou, Agnes Rötig, Aleksandra Filipovska, Ingrid Cristian, Marni J. Falk, Metodi D. Metodiev, David R. Thorburn  The American Journal of Human Genetics  Volume 101, Issue 2, Pages 239-254 (August 2017) DOI: 10.1016/j.ajhg.2017.07.005 Copyright © 2017 American Society of Human Genetics Terms and Conditions

Figure 1 Identification of MRPS34 Mutations in Six Subjects from Four Families (A) Pedigrees and genotype of subjects with MRPS34 variants. Minus sign (−) denotes a mutant allele. (B) Sequencing chromatograms confirming the MRPS34 variants in affected subjects and the carrier status of family members with DNA available. (C) Protein sequence alignment of human MRPS34 with its homologs in nine other vertebrate species showing the conservation of the p.Glu13 residue mutated in family 4. Asterisks (∗) depict conserved amino acids. The American Journal of Human Genetics 2017 101, 239-254DOI: (10.1016/j.ajhg.2017.07.005) Copyright © 2017 American Society of Human Genetics Terms and Conditions

Figure 2 Characterization of MRPS34 in Affected Subjects with MRPS34 Mutations (A) PCR amplicons of MRPS34 exons 1–3 generated from control individual and subject 1 fibroblast cDNA ± cycloheximide (CHX). The amplicon detected in subject 1 was smaller than the control amplicon. (B) Sequence analysis of the MRPS34 cDNA PCR amplicon detected in subject 1 identified a 24 bp deletion corresponding to the utilization of an upstream donor site in exon 1. This splicing mutation therefore produces a shortened transcript that results in an in-frame deletion of eight amino acids, p.Val100_Gln107del. (C) Schematic diagram depicting the abnormal transcript generated from the c.312+1G>T variant (family 1) and two abnormal plus residual wild-type transcript generated from the c.322−10G>A variant (families 2 and 3). The distribution of the three transcripts generated from the c.322−10G>A variant in S2a fibroblasts (40 clones sequenced) and lymphoblasts (40 clones sequenced) is additionally described in the table. The red line indicates the position of the variant. The diagram solid black bars represent exons, while the open bars represent untranslated region. (D) Protein sequence alignment of human MRPS34 with its homologs in nine other vertebrate species. Asterisks (∗) depict conserved amino acids. The eight amino acids missing from the MRPS34 protein produced in subject 1 are highly conserved across the species examined. (E) SDS-PAGE western blot of MRPS34 and complex II 70 kDa subunit SDHA (loading control) from control individuals (C1 and C2) and subject 1 fibroblasts and liver showed the absence of wild-type MRPS34 protein in subject 1. Long exposures revealed faint double banding in subject 1 fibroblast samples probed with MRPS34 antibody. (F and G) SDS-PAGE western blot of MRPS34 in fibroblasts and lymphoblasts revealed a substantial decrease in MRPS34 levels in subjects 2a (F), 2b (F), and 4 (G) relative to control individuals (C1 and C2) and to parental samples (I-2 and I-1 from family 2). Complex II subunits SDHA and SDHB, VDAC1, and citrate synthase represent loading controls. The American Journal of Human Genetics 2017 101, 239-254DOI: (10.1016/j.ajhg.2017.07.005) Copyright © 2017 American Society of Human Genetics Terms and Conditions

Figure 3 Evidence of Combined OXPHOS Deficiency and Reduced Mitochondrial Translation in Affected Subjects with MRPS34 Mutations (A–C) SDS-PAGE western blot of protein from fibroblasts and lymphoblasts showed reduced levels of complex I (CI) and complex IV (CIV) subunits in subjects 1 (A), 2a (B), 2b (B), and 4 (C) relative to control individuals (C1–C3) and to parental samples (I-2 and I-1 from family 2). Complex II subunits (SDHA and SDHB) are indicative of loading. (D and E) BN-PAGE western blot of fibroblast protein showed reduced levels of CI and CIV in subjects 1 (D) and 4 (E) relative to control individuals (C, C1, and C2). Complex II (SDHA and SDHB) is indicative of loading. (F and G) Protein synthesis in cell lysates was measured by pulse incorporation of 35S-labeled methionine and cysteine. Equal amounts of cellular protein were separated by SDS-PAGE and visualized by autoradiography. The in vitro pulse labeling of mitochondrial translation products revealed decreased levels of mtDNA-encoded subunits in subject 1 (F) and subject 4 (G) relative to control individuals (C1 and C2). The Coomassie stain represents relative loading. (H) Examination of mitochondrial protein synthesis in control individual (C) and subject 1 fibroblasts by [35S]-methionine radiolabelling. Isolated mitochondria were subject to BN-PAGE, following which the complexes were visualized by autoradiography. A slower formation of complex IV was observed in subject 1 relative to control individual. SDHA was used as a loading control. Asterisk (∗) denotes a non-specific band. The American Journal of Human Genetics 2017 101, 239-254DOI: (10.1016/j.ajhg.2017.07.005) Copyright © 2017 American Society of Human Genetics Terms and Conditions

Figure 4 MRPS34 Mutations Are Associated with Reduced Protein Levels of Small Mitoribosomal Subunits and Destabilization of the Mitoribosome (A and B) SDS-PAGE western blot of protein from fibroblasts showed reduced protein levels of small mitoribosomal subunit proteins in subjects 1 (A) and 4 (B) relative to control individuals (C1 and C2). The abundance of large mitoribosomal proteins in subjects 1 and 4 were comparable to control individuals. Complex II subunit SDHA, VDAC1, and GAPDH were used as loading controls. (C) A continuous 10%–30% sucrose gradient was used to determine the distribution of the small and large ribosomal subunit and the monosome in mitochondria isolated from control individual (C) and subject 1 cells. Mitochondrial ribosomal protein markers of the small (MRPS16 and MRPS35) and large (MRPL11 and MRPL37) ribosomal subunits were detected by immunoblotting with specific antibodies. The data are representative of results from three independent biological experiments. The dashed vertical lines denote the relevant fractions as indicated. The American Journal of Human Genetics 2017 101, 239-254DOI: (10.1016/j.ajhg.2017.07.005) Copyright © 2017 American Society of Human Genetics Terms and Conditions

Figure 5 Quantitative Proteomic Analysis of Fibroblasts from an Affected Subject with MRPS34 Mutations Identifies a General Decrease in Small Mitoribosomal and OXPHOS Subunit Proteins (A) Quantitative mass spectrometry of mitochondrial proteins in fibroblasts from subject 1 and control individuals demonstrates downregulation of small mitoribosome subunits (red dots), as well as OXPHOS subunits (blue dots), in subject 1. In contrast, the levels of large mitoribosome subunits (yellow dots) in subject 1 are generally unaffected. Proteins examined in this study by SDS-PAGE and observed to have reduced levels are indicated by the text labels. The horizontal line within the volcano plot represents a significance value of p = 0.05, where the levels of proteins represented above the horizontal p = 0.05 line was regarded as significantly different from control individuals. The two dashed vertical lines indicate Log2 changes of >0.5 up- or downregulation relative to control individuals. (B) OXPHOS and mitoribosome protein levels in subject 1 represented as a ratio of the control mean. Hashes denote groups that were significantly reduced in subject 1 relative to control individuals (all with p value < 0.0001). The middle bar represents the mean value, while the upper and lower bars represent the 95% confidence interval of the mean value. Each dot represents a single protein. (C) Changes in mitoribosome protein levels between subject 1 relative to control individuals mapped to the structure of the human mitoribosome. As per the inset scale, proteins colored in blue are decreased, and those colored in red are increased, in subject 1 relative to control individuals. Grey indicates no data; yellow indicates MRPS34. (D) Changes in OXPHOS subunit levels for complexes I–IV mapped to homologous subunits of the relevant structure. Color scale as per (C). The American Journal of Human Genetics 2017 101, 239-254DOI: (10.1016/j.ajhg.2017.07.005) Copyright © 2017 American Society of Human Genetics Terms and Conditions

Figure 6 Lentiviral-Mediated Expression of Wild-Type MRPS34 Rescues the Defect in Mitochondrial Translation in Cells from Affected Subjects (A) Fibroblasts from control individual, subject 1, and a subject with pathogenic MRPS7 variants were transduced with wild-type MRPS34 cDNA. Representative SDS-PAGE western blot demonstrates an increase in protein levels of CI (NDUFB8) and CIV (COXII) subunits in subject 1 fibroblasts transduced with MRPS34 relative to untransduced cells. VDAC1 was used as a loading control. (B and C) Densitometry analysis revealed that the increase in CI subunit NDUFB8 (B) and CIV subunit COXII (C) observed in subject 1 fibroblasts transduced with MRPS34 relative to untransduced cells was significant (p = 0.0049 and 0.036, respectively). Results were normalized to VDAC1 and presented as the percent of average untransduced control cells. The data represent the mean of three independent transfections ± SEM. (D and E) Complex I (D) and complex IV (E) activity was measured in fibroblasts from control individual, subject 1, and a subject with pathogenic MRPS7 variants that were transduced with wild-type MRPS34 cDNA. Complex I and complex IV activity was significantly increased in subject 1 cells transduced with wild-type MRPS34 relative to untransduced cells (both p < 0.0001). Complex IV was significantly decreased in control individual cells transduced with wild-type MRPS34 relative to untransduced cells (p = 0.0004). Results were normalized to citrate synthase and presented as the percent of average untransduced control cells. The data represents the mean of three independent transfections ± SEM. (F) The level of small mitoribosomal subunit proteins MRPS5 and MRPS18B was examined in fibroblasts from control individuals (C1 and C2) and subject 4 transduced with either RFP or wild-type MRPS34 by SDS-PAGE western blotting. Fibroblasts from subject 4 that had been transduced with wild-type MRPS34 had increased levels of MRPS5 and MRPS18B relative to cells transduced with RFP. β-actin was used as a loading control. The blot shown is representative of three independent experiments. The American Journal of Human Genetics 2017 101, 239-254DOI: (10.1016/j.ajhg.2017.07.005) Copyright © 2017 American Society of Human Genetics Terms and Conditions