The Mitochondrial-Encoded Peptide MOTS-c Translocates to the Nucleus to Regulate Nuclear Gene Expression in Response to Metabolic Stress

Abstract

Cellular homeostasis is coordinated through communication between mitochondria and the nucleus, organelles that each possess their own genomes. Whereas the mitochondrial genome is regulated by factors encoded in the nucleus, the nuclear genome is currently not known to be actively controlled by factors encoded in the mitochondrial DNA. Here, we show that MOTS-c, a peptide encoded in the mitochondrial genome, translocates to the nucleus and regulates nuclear gene expression following metabolic stress in a 5'-adenosine monophosphate-activated protein kinase (AMPK)-dependent manner. In the nucleus, MOTS-c regulated a broad range of genes in response to glucose restriction, including those with antioxidant response elements (ARE), and interacted with ARE-regulating stress-responsive transcription factors, such as nuclear factor erythroid 2-related factor 2 (NFE2L2/NRF2). Our findings indicate that the mitochondrial and nuclear genomes co-evolved to independently encode for factors to cross-regulate each other, suggesting that mitonuclear communication is genetically integrated.

Keywords: MOTS-c; homeostasis; metabolic stress; mitochondria; mitochondrial DNA; mitochondrial-derived peptide (MDP); mitonuclear communication; peptide; short open reading frames (sORF); stress response.

Figure 1.. MOTS-c Is a Mitochondrial-Encoded Peptide that Can Reside in the Nucleus

(A) Immunoblots of endogenous MOTS-c from different subcellular fractions of resting HEK293 cells. W, whole-cell lysate; C, cytoplasm; N, nucleus; and M, mitochondria. (B) Endogenous MOTS-c detection by immunofluorescence microscopy in resting HEK293 cells. The nucleus is outlined by white dashed lines. (C) Immunoblot detection of endogenous MOTS-c in increasing levels of nuclear extracts from resting HEK293 cells. Antibody specificity to MOTS-c was confirmed by neutralizing peptide competition. Nuclear proteins such as lamin B1 and histone H2B were used as nuclear loading controls. (D) Localization of exogenously treated FITC-MOTS-c peptide (1 μM; 30 min) in HEK293 cells by confocal microscopy. (E and F) Subcellular distribution pattern of wild-type (WT) and mutant (₈YIFY₁₁ → ₈AAAA₁₁ and ₁₃RKLR₁₆ → ₁₃AAAA₁₆) MOTS-c tagged with EGFP in resting HEK293 cells by (E) confocal microscopy (EGFP-MOTS-c^WT, EGFP-MOTS-c^YIFY, and EGFP-MOTS-c^RKLR, respectively) and (F) immunoblotting of subcellular fractions (M^WT, M^YIFY, and M^RKLR, respectively). Representative images are shown (n = 3). Cells were co-stained with DAPI (nucleus) and MitoTracker Red (mitochondria). Scale bar, 10 μm. See also Figure S1.

Figure 2.. Metabolic Stress Triggers MOTS-c to Dynamically Translocate into the Nucleus

(A–G) Spatial and temporal assessment of MOTS-c localization in HEK293 after glucose restriction (GR; 0.5 g/L), serum deprivation (SD; 1% fetal bovine serum), and tert−butyl hydrogen peroxide (tBHP; 100 μM) by (A–C) subcellular fraction immunoblots and (D–G) immunofluorescence microscopy. Subcellular fractions were purified at 0, 0.5, 1, 3, 6, and 24 hr post-stress, and confocal microscopy images were acquired 3 hr post-stress. Representative images shown (n = 3). Cells were co-stained with DAPI (nucleus) and MitoTracker Red (mitochondria). Scale bar, 10 μm. (H) Flow cytometry based on DHE to assess time-dependent reactive oxygen species (ROS) production in response to GR (0.5 g/L), SD (1% fetal bovine serum), and tBHP (100 μM) in HEK293 cells (n = 4). (I) Fluorescence-activated cell sorting based on MitoSOX staining to assess mitochondrial ROS production following control (DMSO; 0.05%), rotenone (Rot; 10 μM), GR (0.5 g/L), SD (1% fetal bovine serum), and tBHP (100 μM) in HEK293 cells (n = 4). (J) Subcellular fraction immunoblots following tBHP (100 μM) treatment with and without pre-treatment with NAC (10 mM) for 2 hr. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test. See also Figure S2.

Figure 3.. Stress-Induced Nuclear Translocation of MOTS-c Is Dependent on AMPK

(A and B) The level of nuclear MOTS-c after 1 hr of GR, SD, and/or tBHP (100 μM) treatment with and without (A) compound C (10 μM), an AMPK inhibitor, for 30 min prior to stress and (B) siRNA against AMPKα or a non-specific sequence (cont) for 48 hr prior to stress, determined by subcellular fractionation immunoblots. (C) Immunofluorescence staining of MOTS-c after metformin (5 mM; left) or AICAR treatment (2 mM; right). (D and E) Immunoblots of nuclear fractions from cells treated with metformin or AICAR with and without (D) compound C (10 μM) or (E) siRNA against AMPKα or a non-specific sequence (cont). Representative images are shown (n = 3). Cells were co-stained with DAPI (nucleus). Scale bar, 10 μm. See also Figure S3.

Figure 4.. MOTS-c Binds to Nuclear DNA and Interacts with Nrf2 to Regulate Gene Expression

(A and B) Immunoblots of endogenous MOTS-c from chromatin extracts of (A) resting HEK293 cells and (B) HEK293 and HepG2 cells with and without GR (3 hr). (C) Co-immunoprecipitation of NRF2 by anti-MOTS-c antibody from HEK293 cell nuclear extracts at 0, 3, and 24 hr after GR (top panel) or tBHP (100 μM; bottom panel) determined by immunoblotting. (D) Co-immunoprecipitation of EGFP-MOTS-c and Myc-tagged NRF2 (Myc- NRF2) in HEK293 cells determined by immunoblotting. (E) MOTS-c^YIFY: ₈YIFY₁₁ → ₈AAAA₁₁ and pMOTS-c^RKLR : ₁₃RKLR₁₆ → ₁₃AAAA₁₆ mutant MOTS-c peptides. Direct MOTS-c/DNA interactions were examined by electrophoretic mobility shift assay (EMSA). (F) ChIP-qPCR analysis of ARE-containing promoter regions of NQO1 (left) and HO-1 (right) bound to MOTS-c at 0, 3, and 24 hr after GR (top panels) and tBHP (100 μM; bottom panels) (n = 3). (G) ChIP-qPCR analysis of ARE-containing promoter regions of HO-1 bound to NRF2 in HEK293 cells transfected with empty vector (pEV) or MOTS-c (pMOTS-c) plasmid (n = 3). (H) ARE-luciferase reporter activity on cells transfected with pEV or pMOTS-c in HEK293 cells in combination with siRNA against NRF2 or a non-specific control (NS) (n = 15). (I) qRT-PCR analysis of HO-1 and NQO1 expression in response to the overexpression of WT or mutant MOTS-c in HEK293 cells (n = 3). (J and K) HEK293 cells were stably transfected with pEV, pMOTS-c^WT, pMOTS-c^YIFY, and pMOTS-c^RKLR and subjected to 96 hr of GR + SD. Survival was assessed by (J) phase contrast microscopy images and (K) flow cytometry (n = 3). (L–N) RNA-seq analyses on HEK293 cells that were transfected with MOTS-c (or empty vector) and subjected to glucose restriction (GR) for 3 hr (n = 6). (L) Heatmap of significantly differentially regulated genes by MOTS-c upon GR at false discovery rate (FDR) < 5%. (M) Overlap of genes upregulated by MOTS-c during GR and bona fide NRF2 target genes. (N) Significantly enriched transcription factor DNA binding motifs in the promoters of genes upregulated (top) and downregulated (bottom) by MOTS-c. (O) Co-immunoprecipitation of MOTS-c and ATF1 from nuclear extracts of HEK293 cells after GR (3 hr) determined by immunoblotting. (P) A schematic illustration of the nuclear role of the mitochondrial-encoded peptide MOTS-c in response to metabolic stress. Error bars represent mean ± SEM. **p < 0.01, ***p < 0.001 by Student’s t test. See also Figure S4 and Tables S1 and S2.