CRISPR/Cas9 screening identifies essential genes in the regulation of mitochondrial function

a, Schematic diagram of screening strategy. Cell populations with sgRNA library were cultured in glucose medium or galactose medium (galactose was used to replace glucose) for 48 hours. Dead cells from galactose medium or living cells from glucose medium were collected to carry out next-generation sequencing for evaluating the abundance of sgRNAs. b, Genes essential for mitochondrial oxidative phosphorylation (OXPHOS) -linked energy metabolism were identified in the screening. A rank-ordered plot shows the RRA (Robust Rank Aggregation) scores and corresponding p-values from the CRISPR screening results. c, Fold change (FC) distribution of sgRNAs targeting Eola1, Sp1, Stat1, and Samhd1 as indicated in red (upregulated) and blue (downregulated) lines, overlaid on gray gradient depicting the overall distribution. d, e, Growth curves of wild-type (WT) and EOLA1 KO cells cultured in the medium of glucose (d) or galactose (e). f, Eola1 depletion dramatically induced cell apoptosis when cancer cells cultured in galactose medium. Representative flow cytometry images (left) and quantification analysis (right) of the apoptotic B16-F10 melanoma cells and HL-1 cells cultured in glucose medium or galactose medium for 48 hours. FITC: fluorescein isothiocyanate; PI: propidium iodide. Note: d, e, One-way ANOVA with Tukey’s test. f, two-tailed unpaired Student’s t-tests. *P<0.05, ****P<0.0001, ns: not significant. d, e, f, Data were presented as mean ± SD (n=3).

EOLA1 is a novel mitochondrial protein and required for mitochondrial activity

a, Mitochondrial proteins predicted by TargetP 2.0 in human and mouse (left), and their overlap with MitoCarta 3.0 annotations (right). b, Fluorescence analysis of the subcellular location of human EOLA1 protein (red), and HSP60 (green) was used as a marker for mitochondria. The exogenously expressed EOLA1 was tagged with a C-terminal FLAG and HA tandem epitope. c, Western blotting analysis of EOLA1 in the subcellular fractions of PLC/PRF/5 (Homo sapiens, left) and B16-F10 melanoma cells (Mus musculus, right). Tubulin was used as a cytoplasmic marker, Histone H1.2 as a nuclear marker, and HSP60 as a mitochondrial marker. d, Determination of EOLA1 sub-mitochondrial localization by Proteinase K digestion assay using purified mitochondria from HEK293T cells. PK, Protease K; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane. e, f, Seahorse analysis of WT and Eola1 KO in B16-F10 melanoma cells (e) and HL-1 cells (f). Quantification of the basal respiration, maximal respiration, and spare respiratory capacity was performed for the indicated cells. g-i, Electron microscopy images (g) of WT cells (left) and Eola1 knockout cells (right). Higher-magnification views are presented in panel h. The relative circularity ratio of mitochondria was quantified (i). Mito: Mitochondria, N: nucleus. Note: e, f, One-way ANOVA with Tukey’s test. i, two-tailed unpaired Student’s t-tests. **P<0.01, ***P<0.001, ****P<0.0001, ns: not significant. e, f, Data were presented as mean ± SD (n=6).

EOLA1 regulates mitochondrial mRNA translation by interacting with TUFM and 12S mt-rRNA

a, b, Identification of EOLA1 interacting partners. EOLA1 complex was purified from EOLA1–FH stable cells using immunoaffinity purification. Eluted proteins were separated by SDS-PAGE and analyzed by silver staining (a). Western blotting analysis verified the mitochondrial Tu translation elongation factor (TUFM) as an EOLA1-binding partner (b). c-e, RNA immunoprecipitation (RIP) followed by qPCR demonstrates specific binding between EOLA1 and 12S mt-rRNA. Exogenous RIP-qPCR with (c) or without (d) UV crosslinking confirms direct RNA-protein interactions. Endogenous RIP-qPCR further validates this physiological association (e). The identification primers are provided in supplementary Table 1. f, g, EOLA1 affects mitochondrial mRNA translation. Sucrose gradient fractionation profiles of mitochondrial ribosomal subunits assessed by RT-PCR of 12S rRNA (mt-SSU) and 16S rRNA (mt-LSU) (f, top). Distribution of mitochondrial-encoded mRNAs (ATP6, ND5, and COX1) measured by RT-PCR (f, bottom), along with their corresponding protein levels measured by Western blotting. β-actin served as a loading control (g). The identification primers are provided in supplementary Table 1. h, Mitochondrial translation activity was assessed using the Mito-Click-iT assay. i, Mitochondrial-encoded mRNAs (mt-mRNAs) and ribosomal RNA levels (12S and 16S mt-rRNA) were quantified in control and EOLA1-knockout cells by RT-qPCR. The identification primers are provided in supplementary Table 1. Note: c, d, e, two-tailed unpaired Student’s t-tests. i, One-way ANOVA with Tukey’s test. ***P<0.001, ****P<0.0001. c, d, e, i, Data were presented as mean ± SD (n=3).

Abnormal cardiac function was observed in Eola1-/- mice

a, EOLA1 protein deficiency in knockout mice was validated by Western blotting analysis. GAPDH was used as a loading control. b, Western blotting analysis revealed significantly lower levels of mitochondrial protein in Eola1-/- hearts compared to Eola1+/+. GAPDH was used as a loading control. c, Typical ventricular borders at end-diastole were identified by B-mode echocardiography. d, e, Compared to Eola1+/+ mice, both male (d) and female (e) Eola1-/- mice demonstrated significant enlargement in ventricular long-axis length (i), short-axis length (ii), and cross-sectional area (iii). f, Representative images of left ventricular motion patterns identified by M-mode echocardiography. g, Compared to Eola1+/+ mice, male Eola1-/- mice demonstrated a significant decrease in ejection fraction (i), shortening fraction (ii), and stroke volume (iv) of left ventricle. Cardiac output (iii) of left ventricle also showed a decreasing trend. h, Compared to Eola1+/+ mice, female Eola1-/- mice demonstrated a significant decrease in ejection fraction (i), shortening fraction (ii), cardiac output (iii) and stroke volume (iv) of left ventricle. i, Eola1 knockout leads to thickening of the anterior wall (i) and thinning of the posterior wall (ii) of the left ventricle in male mice. j, Eola1 deficiency causes thinning of the posterior wall (ii) and a trend of thickening of the anterior wall (i) of the left ventricle in female mice. k, l, Eola1 deficiency increases heart rate of both male (k) and female (l) mice. Note: d, e, g-l, two-tailed unpaired Student’s t-tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns: not significant. Data were presented as mean ± SEM.

Working model illustrating the critical role of mitochondrial protein EOLA1 in heart function

EOLA1, a mitochondrial matrix protein identified by CRISPR screening, interacts with TUFM/12S mt-rRNA to promote the protein synthesis of OXPHOS subunits. Its loss impairs mt-mRNA translation and causes heart failure in mice, revealing a mitochondrial translation-cardiac function axis pivotal for cardiovascular homeostasis.

Generation and validation of Eola1-knockout cell lines

a, Evolutionary analysis of the ASCH domain reveals high conservation across species. b, A scheme showing functional domains of EOLA1 in Homo sapiens (top) and Mus musculus (bottom). The ASCH domain is shown in red. c, Schematic diagram of sgRNA targeting mouse Eola1 locus. d, Western blotting analysis confirming Eola1 knockout clones in B16-F10 and HL-1 cell lines with anti-EOLA1 antibody. GAPDH was used as a loading control. e, Sanger sequencing results of Eola1 KO cell lines with genetic mutations introduced with the CRISPR/Cas9 system.

Mislocalization of EOLA1 upon deletion of its N-terminal targeting signal

a, Evolutionary conservation of EOLA1’s N-terminal domain. Sequence alignment across diverse species reveals key conserved residues (red). b, Fluorescence imaging analysis of EOLA1 subcellular localization after N-terminal mitochondrial targeting signal (MTS) deletion (red). Mitochondria were labeled with TFAM (green). Exogenously expressed MTS-deficient EOLA1 (delMTS-EOLA1) carried a C-terminal FLAG-HA tag.

EOLA1 is associated with mitochondrial translation

a, Pie chart representing the proportion of mitochondrial proteins identified in the EOLA1 interactome. b, EOLA1-interacting mitochondrial RNAs were identified via UV-RIP-seq. c, Protein levels of MRPS15 (mt-SSU marker) and MRPL11 (mt-LSU marker) were analyzed by Western blotting across fractions.

Generation and phenotypic analysis of Eola1-/- mice

a, b, Schematic diagram of sgRNA targeting mouse Eola1 locus (a) and validation of Eola1 knockout in mice by PCR (b). The genotyping primers are provided in supplementary Table 1. c, Representative pictures of the Eola1+/+ and Eola1-/- mice. d, Comparison of body weight in female (left) and male (right) Eola1+/+ and Eola1-/- mice. e, Relative mRNA levels of mitochondrial-encoded transcripts were quantified by RT-qPCR in Eola1+/+ and Eola1-/- hearts. Note: e, two-tailed unpaired Student’s t-tests. ****P<0.0001, ns: not significant. Data were presented as mean ± SD (n=6).

List of PCR primers used in this study