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The Protein Coded by a Short Open Reading Frame, Not The Protein Coded by a Short Open Reading Frame, Not by the Annotated Coding Sequence, Is the Main Gene Product of the Dual-Coding Gene MIEF1 Vivian Delcourt, Mylène Brunelle, Annie Roy, Jean-François Jacques, Michel Salzet, Isabelle Fournier, Xavier Roucou To cite this version: Vivian Delcourt, Mylène Brunelle, Annie Roy, Jean-François Jacques, Michel Salzet, et al.. The Protein Coded by a Short Open Reading Frame, Not by the Annotated Coding Sequence, Is the Main Gene Product of the Dual-Coding Gene MIEF1. Molecular and Cellular Proteomics, American Society for Biochemistry and Molecular Biology, 2018, 17 (12), pp.2402-2411. 10.1074/mcp.RA118.000593. inserm-02940894 HAL Id: inserm-02940894 https://www.hal.inserm.fr/inserm-02940894 Submitted on 16 Sep 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. los Research Author’s Choice The Protein Coded by a Short Open Reading Frame, Not by the Annotated Coding Sequence, Is the Main Gene Product of the Dual-Coding Gene MIEF1*□S Vivian Delcourt‡§¶, Myle`ne Brunelle‡¶, Annie V. Roy‡¶, Jean-Franc¸ois Jacques‡¶, Michel Salzet§, Isabelle Fournier§, and Xavier Roucou‡¶ʈ Proteogenomics and ribosome profiling concurrently some profiling and proteogenomics. Ribosome profiling maps show that genes may code for both a large and one or the regions of the transcriptome which are actively translated Downloaded from more small proteins translated from annotated coding with nucleotide resolution (2). Proteogenomics approaches sequences (CDSs) and unannotated alternative open use customized protein databases and mass spectrometry reading frames (named alternative ORFs or altORFs), re- (MS)-based proteomics to detect translated proteins (3–10). spectively, but the stoichiometry between large and small Both methods have revealed prevalent translation of ORFs proteins translated from a same gene is unknown. MIEF1, a gene recently identified as a dual-coding gene, harbors outside of annotated CDSs and of out-of-frame ORFs https://www.mcponline.org a CDS and a newly annotated and actively translated (altORFs) (2, 11). These findings call into question the concept altORF located in the 5؅UTR. Here, we use absolute quan- of the single CDS in eukaryotic mRNAs (12). In addition, they tification with stable isotope-labeled peptides and parallel also highlight the need to redefine translated sequences (13), reaction monitoring to determine levels of both proteins in modernize functional genome annotations with shorter ORFs two human cells lines and in human colon. We report that (14), and reassess the translation output of protein coding- the main MIEF1 translational product is not the canonical genes by considering smaller proteins in addition to larger 463 amino acid MiD51 protein but the small 70 amino acid canonical proteins. The cellular stoichiometry of a canonical at INSERM on September 17, 2020 alternative MiD51 protein (altMiD51). These results dem- protein versus a small protein encoded in the same gene and onstrate the inadequacy of the single CDS concept and provide a strong argument for incorporating altORFs and their respective concentrations is unknown. Yet, proteins are small proteins in functional annotations. Molecular & the primary effectors of biological processes and deciphering Cellular Proteomics 17: 2402–2411, 2018. DOI: 10.1074/ the function of a gene in health and disease requires accurate mcp.RA118.000593. characterization of its products. The mitochondrial elongation factor 1 gene or MIEF1 also termed SMCR7L/MiD51, localized at the Chr22q13.1 locus, According to the traditional view of protein synthesis, each codes for a mitochondrial receptor of Drp1, a GTPase which protein-coding gene harbors a single annotated open reading functions in mitochondrial fission (15–17). frames (ORF)1 or coding sequence (CDS) encoding a canon- Ribosome profiling and proteogeomics studies recently ical protein. However, genes contain more than one ORF, and demonstrated the translation of a stable 70 amino acid protein the longest ORF is generally designated as the canonical CDS in product encoded in a altORF localized in the 5ЈUTR (Fig. 1A) genome annotations (1). In eukaryotes, alternative splicing re- (3, 18–24). Thus, MIEF1 is a prototypical gene coding for both sults in the production of several mRNAs and the translation of a large and a small protein. For simplicity, we termed this different isoforms, in addition to the canonical protein. Hence, novel protein “alternative-MiD51” or altMiD51. Remarkably, the translational output of a protein-coding gene is currently both proteins are localized at the mitochondria. MiD51 is an concealed to a canonical protein and one or several isoforms. outer mitochondrial membrane protein (17) whereas alt- This concept was recently disproved by two modern ap- MiD51 is located at the mitochondrial matrix (24) and both proaches to the accurate measurement of translation, ribo- are involved in mitochondrial fission (17, 24). AltMiD51 has From the ‡De´partement de Biochimie, Universite´ de Sherbrooke, Que´bec, Canada; §Univ. Lille, INSERM U1192, Laboratoire Prote´omique, Re´ponse Inflammatoire and Spectrome´trie de Masse (PRISM) F-59000 Lille, France; ¶PROTEO, Que´bec Network for Research on Protein Function, Structure, and Engineering, Que´bec, Canada Author’s Choice—Final version open access under the terms of the Creative Commons CC-BY license. Received January 23, 2018, and in revised form, July 19, 2018 Published, MCP Papers in Press, September 4, 2018, DOI 10.1074/mcp.RA118.000593 2402 Molecular & Cellular Proteomics 17.12 © 2018 Delcourt et al. Published by The American Society for Biochemistry and Molecular Biology, Inc. Stoichiometry of Two Proteins Coded by a Human Gene also been reported to be a new assembly factor of the mito- containing the altMiD51 coding sequence with a C-terminal 2 FLAG chondrial ribosome and implicated in its biogenesis (25). tag and the canonical MiD51 coding sequence with a C-terminal HA tag (transcript NM_019008.4) into pcDNA 3.1(-) expression vector. Here, we employ a targeted proteomics approach based on DNA sequences were controlled by sequencing. AQUA peptides to reliably quantify the absolute amount of Mitochondrial Extracts—For Western blot analysis of HeLa and MiD51 and altMiD51 in two human cell lines and one human CRISPR-Cas9 HeLa clones as well as PRM optimizations, mitochon- tissue, and thus we establish an improved map of the trans- drial extracts were performed according to (28) with minor modifica- lational output of MIEF1/SMCR7L/MiD51 by directly meas- tions. Cells were grown into three 100 mm dishes until 80% confluent, rinsed twice with PBS 1ϫ and collected using a cell scrapper. Cells uring the final protein products. were pelleted by centrifugation at 500 ϫ g for 10 min at 4 °C. Super- natant was discarded and cells were suspended in mitochondrial EXPERIMENTAL PROCEDURES buffer (mito-buffer : 210 mM mannitol, 70 mM sucrose, 1 mM EDTA, 10 Experimental Design and Statistical Rationale—In this study, our mM HEPES-NaOH, pH 7.5, 2 mg/ml Bovine Serum Albumin (BSA), 0.5 aim is to determine the stoichiometry of two distinct proteins encoded mM PMSF and EDTA-free protease inhibitor (Thermo Fisher Scien- within the gene MIEF1, the canonical protein MiD51 and altMiD51. tific)) and disrupted by passage through a 25G1 0.5 ϫ 25 needle AltMiD51 is a 70 amino acids protein coded by a short ORF in the syringe 15 consecutive times on ice followed by a 3 min centrifugation 5ЈUTR region. We expect to unveil the stoichiometry of the two at 2000 ϫ g at 4 °C. Supernatant was collected and the pellet was proteins using targeted proteomics and stable isotope labeled syn- resuspended in mitochondrial buffer. The breakage procedure was thetic peptides. To determine proteotypic peptides, we first per- repeated four times. All four supernatants containing mitochondria formed AP-MS DDA experiments on both proteins. Peptides were were again passed through syringe needle in mito-buffer and cleared Downloaded from then validated in overexpressed and endogenous conditions using by centrifugation for 3 min at 2000 ϫ g at 4 °C. Supernatants were targeted proteomics. Selected peptides were tested for coefficient of collected and centrifuged for 10 min at 13,000 ϫ g at 4 °C to pellet variation (CV) using technical replicates and were validated if their CV mitochondria. Pellets were washed twice with BSA-free mitochondrial fell below 20%. One stable isotope peptide for each protein was buffer and pooled. Final mitochondrial pellet was lysed in SDS buffer purchased and tested for linearity in a peptide mixture using two (4% SDS, Tris-HCl 100 mM pH 7.6). After sonication, protein content technical replicates at various concentrations. Both peptides showed was assessed using BCA assay (Thermo Fisher Scientific). https://www.mcponline.org a linearity range between 40 amol and 250 fmol. Absolute quantifica- tion experiments were then conducted in two cell lines (three biolog- Mass Spectrometry Sample Preparation ical replicates each) and colon tissue (three technical replicates), in MiD51 knockout HeLa cells and altMiD51 knockout HeLa cells (three Preliminary Affinity-purification (AP)—Cells were rinsed twice with biological replicates each). Protein amounts were compared using cold PBS 1X and lysed with 1 ml of AP-buffer (NP-40 0.5%, Tris-HCl Welch’s two samples t-tests. 50 mM pH 7.5, NaCl 150 mM, EDTA-free protease inhibitor 1ϫ). Lysate Tissue Collection and Ethics—Normal human colon tissue sample was cleared by centrifugation (2000 ϫ g, 5 min) and supernatant was was obtained from the Biobanque des maladies digestives du collected.
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