Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency

Abstract

Mitochondria are composed of many small proteins that control protein synthesis, complex assembly, metabolism, and ion and reactive oxygen species (ROS) handling. We show that a skeletal muscle- and heart-enriched long non-coding RNA, LINC00116, encodes a highly conserved 56-amino-acid microprotein that we named mitoregulin (Mtln). Mtln localizes to the inner mitochondrial membrane, where it binds cardiolipin and influences protein complex assembly. In cultured cells, Mtln overexpression increases mitochondrial membrane potential, respiration rates, and Ca²⁺ retention capacity while decreasing mitochondrial ROS and matrix-free Ca²⁺. Mtln-knockout mice display perturbations in mitochondrial respiratory (super)complex formation and activity, fatty acid oxidation, tricarboxylic acid (TCA) cycle enzymes, and Ca²⁺ retention capacity. Blue-native gel electrophoresis revealed that Mtln co-migrates alongside several complexes, including the complex I assembly module, complex V, and supercomplexes. Under denaturing conditions, Mtln remains in high-molecular-weight complexes, supporting its role as a sticky molecular tether that enhances respiratory efficiency by bolstering protein complex assembly and/or stability.

Keywords: ROS; assembly factor; fatty acid oxidation; micropeptide; microprotein; mitochondria; mitochondrial calcium; respirasome; sORF; supercomplexes.

Figure 1.. Human LINC00116 Encodes a Highly Conserved Muscle- and Heart-Enriched Microprotein

(A) Summarized LINC00116 RNA expression data from four independent human RNA-seq body-map datasets are plotted (mean ± SEM) relative to skeletal muscle, which had the highest LINC00116 expression levels in all four datasets. (B) Schematic of the LINC00116 locus genomic architecture based on UCSC Genome Browser (hg19) annotations and expression data from GWIPS-viz ribosomal profiling database, the latter of which indicates the presence of a highly conserved sORF that is translated by ribosomes (ribo-seq). (C) Multi-species alignment of the predicted LINC00116-derived microprotein (NC116-MP) performed using PRALINE algorithm after in silico translation of the conserved homologous sORFs in the indicated species. A conserved transmembrane (TM) domain is predicted in all species (PHOBIUS and TMHMM algorithms). The majority of non-perfectly conserved residues (red) represent conservative substitutions (higher PRALINE scores, range 0–10). (D) A custom antibody was generated against the indicated C-terminal region, and western blot analysis on mouse tissue panels revealed a prominent 10-kDa band enriched in muscle and heart tissues. This band was not present in NC116-MP knockout mouse tissues (Figure S1). (E) RNA co-expression analysis done by querying CoXpresDB database for LINC00116 and the mouse homolog, 1500011K16Rik indicated a clear overlap of co-expressed genes in both species. Gene ontology analyses revealed that the overlapping genes are strongly enriched for mitochondrial-related functions (Table S2). Among the 85 shared genes, 34 have reported functions related to mitochondrial complex and supercomplex assembly (Table S3).

Figure 2.. The LINC00116-Derived Microprotein Mitoregulin Localizes to Inner Mitochondrial Membranes and Binds Cardiolipin

(A and B) Wild-type (A) and GFP-tagged (B) human Mtln were expressed in neonatal rat cardiomyocytes, and co-localization with MitoTracker red was evaluated. Representative photomicrographs are shown. Scale bars, 10 μm. (C) Mitochondrial pellets were isolated from wild-type (WT) or Mtln-knockout (KO) C2C12 myoblast cells, and western blot was performed on various fractions. (D) Mitochondrial pellets harvested from WT or Mtln-KO skeletal muscle tissues were treated with increasing digitonin concentrations to release OMMs, and pellet and supernatant fraction fractions were subjected to western blot analysis. Cox4 and Vdac1 are known IMM and OMM proteins, respectively. Gapdh is a cytosolic protein known to associate with mitochondria in some cases. (E) Mitochondrial pellets harvested from WT skeletal muscle tissues were resuspended in isotonic, hypotonic, or isotonic plus triton buffers in the absence or presence of proteinase K and subjected to western blot analysis. Proteins with known localization to various mitochondrial compartments (e.g., matrix, IMM, and intermembrane space [IMS]) were evaluated as controls. (F) Western blot analysis performed on WT and Mtln-KO cardiac tissue lysates subjected to pull-down assay using cardiolipin (CL)-coated or control beads. Subunit c, a known cardiolipin-binding protein, serves as the positive control. (G) Lipid-strip binding assay performed using synthetic Mtln protein followed by anti-Mtln immunoblot.

Figure 3.. Mtln Overexpression Alters Mitochondrial Membrane Potential, ROS, Respiration, and Ca²⁺ Handling in Human HeLa Cells

Doxycycline (DOX)-inducible expression vectors were used to overexpress Mtln and beta-galactosidase (βgal, control) in cultured human HeLa cells, and protein measures and mitochondria-related analyses performed 48 hr later. Mtln-Dox (i.e., no Dox) serves as additional control. (A) Top, western blot shows clear Mtln overexpression in Mtln+Dox cells, relative to controls, but does not significantly alter respiratory complex levels by OXPHOS cocktail blot or BN-PAGE/Coomassie blue (bottom). Bottom, complex I (CI) in-gel activity assay. (B) Representative photomicrographs depicting TMRE (mitochondrial membrane potential) and MitoSOX (ROS) probe intensities in treated cells. Scale bar, 20 μm. Quantified probe intensities are plotted at right. (C) Standard mitochondrial respiration assays were performed in treated cells and oxygen consumption rates (OCR) plotted at right. Refer to Figure S3 for additional measures. (D and E) Mitochondrial Ca²⁺ (mCa²⁺) dynamics were evaluated in treated cells; representative traces and quantified data are shown for Ca²⁺ retention capacity (CRC) assay (D) and measures of matrix-free Ca²⁺ (E). Graphs are plotted as mean ± SEM, sample n is indicated within each bar, and p values were determined by one-way ANOVA with Dunnett’s post hoc (*p < 0.05, **p < 0.01, and ***p < 0.001 compared to βgal+Dox).

Figure 4.. Mtln Suppression Alters Mitochondrial Membrane Potential and ROS in Human HeLa Cells

Mtln expression was inhibited in cultured human HeLa cells using siRNAs, and protein measures and mitochondrial imaging analyses were performed 48 hr later. (A) Western blot shows clear reductions in Mtln expression in cells treated with Mtln-targeted siRNAs (siMtln1–4, each representing unique siRNA sequences), relative to two non-targeted negative control siRNAs (siNC1–2). 10 nM (top) and 50 nM (bottom) doses were tested. OXPHOS cocktail protein levels did not change in response to Mtln knockdown in any siMtln treatment group. (B) Top, representative photomicrographs depicting TMRE (mitochondrial membrane potential) and MitoSOX (ROS) probe intensities in HeLa cells treated with 50 nM of the indicated siRNAs. Scale bar, 20 μm. Bottom, quantified probe intensities are plotted as mean ± SEM, sample n is indicated within each bar, and p values were determined by one-way ANOVA with Dunnett’s post hoc (****p < 0.0001 compared to βgal+Dox).

Figure 5.. Mtln-KO Mice Exhibit Alterations in Mitochondrial Metabolism, Ca²⁺ Retention, and Protein Complex Assemblies

(A) Permeabilized muscle fibers (cardiac, left ventricular [LV], and skeletal muscle [SKM], gastrocnemius) were harvested from fed or fasted (24 hr) WT or Mtln-KO mice, and oxygen consumption rates (OCR) were measured during sequential addition of palmitoyl-carnitine/malate (PC + Mal), 1 mM ADP (Max ADP), and oligomycin; data are plotted as mean ± SEM. Fed: WT n = 7 (4 males, 3 females), KO n = 7 (3 males, 4 females); fasted: n = 4 females for both WT and KO. (B) Mitochondrial Ca²⁺ retention capacities were measured in permeabilized LV fibers from fasted female mice (n = 4/genotype) and plotted as mean ± SEM; **p = 0.01. (C) In-gel CI activity assay was performed on cardiac tissue mitochondrial lysates from fed WT female mice (n = 3–4/genotype, representative gels shown) (left). Red and green arrows denote bands with decreased or increased (respectively) CI activity in KO mice. Top red arrow points to a doublet band in WT hearts, with the top band virtually absent in KO mice. BN-PAGE and western blot for CI subunit NDUFA9 was performed on the same samples (right). See Figure S7B for SKM tissue data. (D) Representative BN-PAGE western blot on cardiac tissue mitochondrial lysates shows co-migration of Mtln with various prominent mitochondrial complexes; see Figure S7D for SKM data. Some non-specific bands (e.g., in the KO lane) arise from the secondary antibody. (E) SDS-PAGE (top, cardiac tissue lysates) and BN-PAGE (bottom, cardiac mitochondrial lysates) western blots for proteins involved in the TCA cycle (OGDH), CI assembly (ACAD9), and FAO (ACAD9 and VLCAD). Arrow indicates ACAD9 dimers. n = 3–4; representative blots are shown. (F) IDPR prediction was run on the Mtln protein sequence (56 amino acids) using the three indicated independent algorithms and output scores plotted, along with the mean score (red). Scores above 0.5 indicate predicted IDPRs (orange). (G) SDS-PAGE western blot for Mtln-containing high-molecular-weight (HMW) assemblies in WT and Mtln-KO mouse tissue panels. Nonspecific band provides loading control for comparing WT and KO lanes.