Infantile encephaloneuromyopathy and defective mitochondrial translation are due to a homozygous RMND1 mutation - PubMed (original) (raw)

. 2012 Oct 5;91(4):729-36.

doi: 10.1016/j.ajhg.2012.08.019. Epub 2012 Sep 27.

Mario H Barros, Simone Sanna-Cherchi, Valentina Emmanuele, Hasan O Akman, Claudia C Ferreiro-Barros, Rita Horvath, Saba Tadesse, Nader El Gharaby, Salvatore DiMauro, Darryl C De Vivo, Aly Shokr, Michio Hirano, Catarina M Quinzii

Affiliations

Infantile encephaloneuromyopathy and defective mitochondrial translation are due to a homozygous RMND1 mutation

Beatriz Garcia-Diaz et al. Am J Hum Genet. 2012.

Abstract

Defects of mitochondrial protein synthesis are clinically and genetically heterogeneous. We previously described a male infant who was born to consanguineous parents and who presented with severe congenital encephalopathy, peripheral neuropathy, myopathy, and lactic acidosis associated with deficiencies of multiple mitochondrial respiratory-chain enzymes and defective mitochondrial translation. In this work, we have characterized four additional affected family members, performed homozygosity mapping, and identified a homozygous splicing mutation in the splice donor site of exon 2 (c.504+1G>A) of RMND1 (required for meiotic nuclear division-1) in the affected individuals. Fibroblasts from affected individuals expressed two aberrant transcripts and had decreased wild-type mRNA and deficiencies of mitochondrial respiratory-chain enzymes. The RMND1 mutation caused haploinsufficiency that was rescued by overexpression of the wild-type transcript in mutant fibroblasts; this overexpression increased the levels and activities of mitochondrial respiratory-chain proteins. Knockdown of RMND1 via shRNA recapitulated the biochemical defect of the mutant fibroblasts, further supporting a loss-of-function pathomechanism in this disease. RMND1 belongs to the sif2 family, an evolutionary conserved group of proteins that share the DUF155 domain, have unknown function, and have never been associated with human disease. We documented that the protein localizes to mitochondria in mammalian and yeast cells. Further studies are necessary for understanding the function of this protein in mitochondrial protein translation.

Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

Pedigree of the family The following symbols are used: black symbols, affected individuals; open symbols, unaffected relatives; small black symbols, stillborns; diagonal lines, deceased persons; and asterisks, DNA available.

Figure 2

Figure 2

Schematic Representation of RMND1 Wild-Type and Aberrant Transcripts RMND1 has four splicing variants, of which only three encode proteins: ENST00000367303 (12 exons, isoform 1 [I1]), ENST00000367303 (11 exons, isoform 2 [I2]), and ENST00000444024 (9 exons, isoform 3 [I3]). All three coding variants contain a DUF155 domain indicated by a purple box. Coding regions are represented by blue boxes, and noncoding regions are represented by white boxes. The longest transcript has a predicted N-terminal 33 amino acid mitochondrial targeting sequence (MTS). On the left is agarose gel showing the PCR product of RMND1 cDNA from control (C) and VI-3 fibroblasts. “L” indicates the 100 bp DNA ladder. Splice-site mutation c.504+1G>A (arrow) produces two aberrant transcripts (AT1 and AT2) and reduces the amount of wild-type transcript (middle band). The longer transcript, AT1, includes the insertion of 88 nucleotides after exon 2 and generates a premature stop at codon 171. In the shorter variant, AT2, a cryptic splice site in exon 2 is activated and results in an in-frame deletion of 75 nucleotides and a protein 25 amino acids shorter than the wild-type. RNA was extracted by the PureLink RNA Mini Kit (Ambion, Austin, TX) and reverse transcribed into cDNA with the VILO RT-PCR kit (Invitrogen, Grand Island, NY). RT-PCR of exons 2–7 was performed, and the RT-PCR products were electrophoresed in 2% agarose gels.

Figure 3

Figure 3

RMND1 Is Partially Reduced in VI-3 Fibroblasts (A) qRT-PCR was performed for characterizing the expression level of RMND1 mRNA in VI-3 fibroblasts with the use of TaqMan Assays for RMND1 and ACTB (β-actin) transcripts (Applied Biosystems). Values are expressed as percentages of controls. Data are represented as the mean ± standard deviation and are the results of at least three different experiments. The asterisk (∗) indicates a Student’s t test p < 0.05. (B and C) Immunoblot for measuring the level of RMND1 in VI-3 fibroblasts. Proteins were extracted, and concentrations were measured with the BCA Protein Assay Kit (Pierce). Five micrograms of protein was electrophoresed in a SDS-12%-PAGE gel, transferred to Immun-Blot PVDF membranes (Biorad, Hercules, CA, USA), and probed with rabbit polyclonal RMND1 antibody (product #HPA031399, Sigma-Aldrich, St. Louis, MO) at a 1:1,000 dilution. A mouse monoclonal actin antibody (Sigma-Aldrich) was used at a 1:10,000 dilution. Protein-antibody interaction was detected with peroxidase-conjugated goat anti-mouse IgG antibody (1:5,000) (Sigma-Aldrich) with SuperSignal chemiluminiscence detection kit (Thermo Fisher Scientific, Waltham, MA). Quantitation of the bands was performed by densitometric analysis with the National Institutes of Health ImageJ software package (version 1.45). Values are expressed as percentages of controls. Data are represented as the mean ± standard deviation and are the results of at least three different experiments. The asterisks (∗) indicate a Student’s t test p < 0.05. The following abbreviations are used: C1–C4, controls; and actin, β-actin.

Figure 4

Figure 4

Complementation Analysis in VI-3 and Control Fibroblasts After transient transfection with a construct encoding wild-type RMND1, mutant cells showed increased biochemical activities of mitochondrial respiratory-chain enzymes normalized to citrate synthase (CS) (A) and an increase in the steady-state level of mitochondrial OXPHOS subunits (B and C). Values are represented as the mean ± standard deviation, reflect the results of at least three different transfection experiments, and are expressed as percentages of the untransfected control. The asterisks (∗) indicate a Student’s t test p < 0.05.

Figure 5

Figure 5

Effects of RMND1 Knockdown in HeLa Cells HeLa cells were cultured in DMEM with 10% fetal bovine serum (FBS) until they were 70%–80% confluent. Transfection with a scramble shRNA-pLKO plasmid (negative control) and a _RMND1_-specific TRC shRNA-pLKO plasmid construct (TRCN0000135730, Sigma Aldrich) was mediated by Lipofectamine 2000 (Invitrogen). Five hours after transfection, cells were selected with Puromycin in DMEM 2% FBS, and transfected clones were individually expanded in DMEM 10% FBS. RMND1 knockdown was assessed in 30 clones by qRT-PCR and immunoblot analysis. (A) Activity of mitochondrial respiratory-chain enzymes. (B and C) Immunoblot analysis of the steady-state levels of mitochondrial respiratory enzymes (B) and pulse labeling with [35S] methionine of mitochondrial proteins (C). A total of 5 × 105 cells cultured in 10 cm culture plates were washed in methionine-free DMEM and subsequently incubated in the same medium supplemented with 15% dialyzed FBS, 1.2 mM sodium pyruvate, and glucose for 30 min. Cytosolic protein synthesis was inhibited by the addition of emetine (0.1 μg/μl) for 7 min at 37°C. Mitochondrial proteins were labeled with 50 μCi [35S]-methionine Redivue (Amersham Biosciences, Piscataway, NJ) in methionine-free medium and incubated for 1 hr at 37°C. After treatment, cells were incubated for 10 min in DMEM supplemented with 10% FBS and were collected by scraping. Protein aliquots (15 μg per sample) were electrophoresed in a 4%–12% Bis-Tris polyacrylamide gradient gel for 4 hr at 85V. The gel was dried for 60 min at 80°C and analyzed with a phosphorimager. The seven subunits of complex I (ND), three subunits of complex IV (COX), and two subunits of complex V (ATP) are indicated at the left. Values are expressed as percentages of the control and are represented as the mean ± standard deviation. One asterisk (∗) indicates a Student’s t test p < 0.05; ∗∗ indicates a Student’s t test p < 0.01.

Figure 6

Figure 6

RMND1 Localization in Mammalian Cells by Immunohistochemistry and Quantitation by Immunoblot Analysis (A) RMND1 cDNA was amplified from a commercial vector containing the human full-length cDNA with the use of a BamHI-recognition-site-integrated forward primer 5′-AGG ATCCGCCATGCCAGCCACACTCCTCAGAGCCG-3′ and AgeI-recognition-site-integrated reverse primer 5′-CGACCGGTGATTTCATGGTTGGAAGGTGTG-3′. PCR product was digested with BamHI and AgeI and cloned into a pEYFP expression vector in-frame and upstream of the YFP coding sequence. Fidelity of the fusion RMND1-YFP construct in the expression vector was verified by sequencing, and 2.5 μg was used for transiently transfecting human embryonic kidney (HEK) 293T cells as described above. Culture media were supplemented with 0.1 μM MitoTracker Red (Invitrogen) for 30 min before the cells were fixed with 4% formalin PBS for the detection of mitochondria. (B) Purification of mitochondria and cellular fractions was performed as previously described, and immunoblot analysis was performed with primary antibodies anti-RMND1 (1:1,000, Sigma), anti-Complex II (1:5,000, MitoScience, Abcam, Cambridge, MA), anti-APH1a (1:1,000, Abcam), and anti-Vinculin (1:5,000, Sigma-Aldrich, St. Louis, MO) and either peroxidase-conjugated anti-rabbit (1:2,000) or peroxidase-conjugated anti-mouse IgG (1:5,000) as a secondary antibody (Sigma-Aldrich). Cellular fractions were isolated from HeLa cells (25 μg of protein from the total lysate [L]), and equal amounts (6 μg) from each fraction (cytoplasmic fraction [CF], endoplasmatic reticulum [ER], crude mitochondria [CMt], pure mitochondria [PMt], and ER + mitochondria [ERMt]) were loaded onto the gel. The known molecular masses (in kDa) of proteins used for confirming cell-fraction enrichment are indicated in the left margin of the gel. (C) Immunoblot analysis of total HEK 293T cell extracts transfected with YFP-RMND1 fusion protein with the antibody against green fluorescent protein confirmed the specificity of the two bands detected by the RMND1 antibody.

References

    1. Ferreiro-Barros C.C., Tengan C.H., Barros M.H., Palenzuela L., Kanki C., Quinzii C., Lou J., El Gharaby N., Shokr A., De Vivo D.C. Neonatal mitochondrial encephaloneuromyopathy due to a defect of mitochondrial protein synthesis. J. Neurol. Sci. 2008;275:128–132. - PMC - PubMed
    1. Kemp J.P., Smith P.M., Pyle A., Neeve V.C., Tuppen H.A., Schara U., Talim B., Topaloglu H., Holinski-Feder E., Abicht A. Nuclear factors involved in mitochondrial translation cause a subgroup of combined respiratory chain deficiency. Brain. 2011;134:183–195. - PMC - PubMed
    1. Rötig A. Human diseases with impaired mitochondrial protein synthesis. Biochim. Biophys. Acta. 2011;1807:1198–1205. - PubMed
    1. Bykhovskaya Y., Casas K., Mengesha E., Inbal A., Fischel-Ghodsian N. Missense mutation in pseudouridine synthase 1 (PUS1) causes mitochondrial myopathy and sideroblastic anemia (MLASA) Am. J. Hum. Genet. 2004;74:1303–1308. - PMC - PubMed
    1. Fernandez-Vizarra E., Berardinelli A., Valente L., Tiranti V., Zeviani M. Nonsense mutation in pseudouridylate synthase 1 (PUS1) in two brothers affected by myopathy, lactic acidosis and sideroblastic anaemia (MLASA) J. Med. Genet. 2007;44:173–180. - PMC - PubMed

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