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

Abstract

Proteogenomics and ribosome profiling concurrently show that genes may code for both a large and one or more small proteins translated from annotated coding sequences (CDSs) and unannotated alternative open reading frames (named alternative ORFs or altORFs), respectively, but the stoichiometry between large and small proteins translated from a same gene is unknown. MIEF1, a gene recently identified as a dual-coding gene, harbors a CDS and a newly annotated and actively translated altORF located in the 5'UTR. Here, we use absolute quantification with stable isotope-labeled peptides and parallel reaction monitoring to determine levels of both proteins in two human cells lines and in human colon. We report that the main MIEF1 translational product is not the canonical 463 amino acid MiD51 protein but the small 70 amino acid alternative MiD51 protein (altMiD51). These results demonstrate the inadequacy of the single CDS concept and provide a strong argument for incorporating altORFs and small proteins in functional annotations.

Keywords: Absolute quantification; Gene Expression*; Knockouts*; Mass Spectrometry; Mitochondria function or biology; Parallel reaction monitoring; Proteogenomics; Translation*; alternative translation; short ORF.

Fig. 1.

Schematic representation of human MIEF1 RefSeq variant 1 mRNA and altMiD51 and MiD51 proteins. A, Human MIEF1 includes 11 exons (RefSeq, GRCh38.p7). The mRNA variant 1 (NM_019008.5) shown here contains 6 exons. The CDS (blue) is shared between exons 3 to 6. AltMiD51 ORF is localized within exon 2, annotated as a non-coding exon. B, Sequence coverage in AP-MS experiments is represented in light colors. Proteotypic peptides sequence and positions (a.a.) are shown. EAVLSLYR and AISAPTSPTR peptides were selected for absolute quantification. *, known phosphorylated residue (phosphosite.org, (63)).

Fig. 2.

Identification and quantification of altMiD51 and MiD51. A, Extracted fragment-ion transition chromatograms of MiD51 ([AISAPTSPTR+2H]²⁺ → y₆⁺) and altMiD51 ([EAVLSLYR+2H]²⁺ → y₄⁺) peptides in HeLa cells. B, Spectral contrast angle analysis of endogenous peptides (top) and stable isotope labeled synthetic peptides (bottom) extracted from supplemental Fig. S5.

Fig. 3.

CRISPR-Cas9 editing of genomic altMiD51 and MiD51. A, Schematic representation of CRISPR-Cas9 experiments strategy. For clarity, only 5 exons are shown. Alt-MiD51 within exon 2 is shown in red. MiD51 coding sequence (blue), overlaps exons 3, 4 and part of exon 5. B, Genomic sequences around the programmed cut sites in non-edited HeLa cells and corresponding sequences. PAM sites are highlighted in yellow, and the genomic sequences targeted by the guide RNAs are highlighted in green. The programmed cut sites are also shown, at nucleotide 40 in exon 2 and nucleotide 137 in exon 3. C, Genomic sequence around the programmed cut sites in CRISPR-Cas9-edited HeLa cells. In the altMiD51-edited clone, a 1 bp A/T insertion (labeled in red, and red star above the electropherogram) occurred at the cut site. In the MiD51edited clone, a mixture of different sequences are detected 10 nucleotides upstream the programmed cut site (red star), indicating the presence of different alleles. D, Mitochondrial extracts from non-edited, MiD51-edited and altMiD51-edited HeLa cells were lysed and analyzed by Western blotting with antibodies against mtHSP70, MiD51 and custom altMiD51 antibodies, as indicated. E, CRISPR-Cas9 MiD51 knock-out sequence analysis. Sequences are aligned with electropherogram of panel c. * refers to a nonspecific target of MiD51 antibodies (17).

Fig. 4.

Absolute quantification and stoichiometries. A, Absolute quantification of altMiD51 and MiD51 in Colon tissue (technical triplicate), HEK 293, HeLa and CRISPR-Cas9 knock outs (biological triplicates). Error bars = standard deviations. B, Stoichiometry determination based on absolute quantities of altMiD51 and MiD51. Boxplots represent three biological (HEK 293 and HeLa) or technical (Colon) replicates. Welch's t test p < 0.05 (*), < 0.01 (**), < 0.001 (***).