Silencing mitochondrial gene expression in living cells

Abstract

Mitochondria fulfill central functions in metabolism and energy supply. They express their own genome, which encodes key subunits of the oxidative phosphorylation system. However, the central mechanisms underlying mitochondrial gene expression remain enigmatic, and a lack of suitable technologies to target mitochondrial protein synthesis in cells has limited experimental access. We silenced the translation of specific mitochondrial mRNAs in living human cells by delivering synthetic peptide-morpholino chimeras. This approach allowed us to perform a comprehensive temporal monitoring of cellular responses. Our study provides insights into mitochondrial translation, its integration into cellular physiology, and provides a strategy to address mitochondrial gene expression in living cells. The approach can potentially be used to analyze mechanisms and pathophysiology of mitochondrial gene expression in a range of cellular model systems.

Fig. 1. Gene silencing by peptide-morpholino chimeras is more efficient and versatile.

See also fig. S1. (A) Jac1-COX1^1-19 and pCox4-COX1^1-19 were imported into isolated mitochondria from HEK293T cells. After import, mitochondria were re-isolated, subjected to in vitro translation in the presence of [³⁵S]methionine, and newly synthesized polypeptides analyzed by SDS-PAGE followed by digital autoradiography. (B and C) Quantification of in organello COX1 silencing effect by Jac1-COX1^1-19 chimera titration (IC₅₀ ~ 0,5 μM; n=4, mean ± SEM) and pCox4-COX1^1-19 chimera titration (IC₅₀ ~ 0,1 μM; n=4, mean ± SEM). (D and E) Titration of pCox4-ATP8^6-28 chimera effect on protein translation in HEK293T cell mitochondria (as in A) and quantification (IC₅₀ ~ 0,1 μM; n=4, mean ± SEM). ATP6 synthesis was also downregulated, confirming coupled synthesis. (F and G) Quantification of in organello silencing by CYTB (pCox4-CYTB^6-24 chimera, IC₅₀ ~ 0,015 μM; n=4, mean ± SEM) and ND2 (pCox4-ND2^12-29 chimera, IC₅₀ ~ 0,03 μM; n=4, mean ± SEM) in HEK293T mitochondria. (H) Silencing the gene expression of one, two, and three mitochondrial-encoded mRNAs in isolated HEK293T mitochondria. Targeted protein indicated by red dot; ATP6 upon ATP8 silencing indicated by gray dot. (I) In organello silencing effect of chimeras targeting different COX1 mRNA 5’ region. Chimera binding region in the transcript are presented as superscript. (J) Heatmap of mtDNA-encoded mRNAs and rRNAs (RNR1 and RNR2) amounts associated with the ribosome upon treatment with COX1-targeting chimeras from (I), compared to the Control (pCox4) (mean, n=3). Statistical significance was determined by multiple t test using the Holm-Sidak method, with alpha=0.05 (^**, padj<0.01). Non-significant differences are not indicated. Mitochondria isolated from mL45^FLAG-expressing HEK293T cells treated with chimeras used in (I), FLAG-IP was performed, RNA purified from eluate fractions, and analyzed by nanoString. COX1 mRNA binding to the ribosome was significantly reduced after treatment with COX1^1-19 and COX1^19-42. FC, fold change. (K) Schematic representation of chimera’s mechanism of action in mitochondria.

Fig. 2. Silencing of mtDNA-encoded genes in living cells.

See also fig. S2. (A) HEK293T cells were transfected with freshly synthesized pCox4-COX1^1-19 at indicated concentrations. No chimera was added to the control (0). 24, 48, and 72 hours post transfection, mitochondrial translation products were metabolically labeled with [³⁵S]methionine. COX1 signal indicated by arrowhead. Steady-state levels of COX1 in cell lysate were determined by immunoblot (IB). (B) HEK293T cells were transfected with pCox4-COX1^1-19 for 3, 6, 16, and 24 hours and silencing confirmed as in (A). COX1 signal is indicated by an arrowhead. (C) Silencing effect of different chimeras targeting COX1 mRNA 5’ region in living cells. Chimera binding region in the transcript presented as superscript. (D) Chimera targeting all mtDNA-encoded genes (except ND6) were transfected in HEK293T cells for 24 hours, followed by [³⁵S]methionine labeling of mitochondrial translation products. Targeted protein indicated by red dot; ATP6 upon ATP8 silencing indicated by gray dot. (E) Simultaneous silencing of two mtDNA-encoded mRNAs in HEK293T cells. (F) Heatmap representing the amount of mtDNA-encoded mRNAs (mt-mRNA) and rRNAs (mt-rRNA, RNR1 and RNR2) associated to the mitochondrial ribosome upon treatment with COX1-, COX2-, and CYTB-targeting chimeras compared to control (pCox4). mL45^FLAG-expressing HEK293T cells were treated with chimeras silencing COX1, COX2, and CYTB for 4, 16, and 16 hours, respectively. Ribosomes were purified by FLAG-IP and associated RNAs detected in the eluate by nanoString (mean, n=3). FC, fold change. Statistical significance was determined by multiple t test using the Holm-Sidak method, with alpha=0.05 (^*, padj<0.05). Non-significant differences are not indicated. (G) Downregulation of mitochondrial gene expression in human iPSC-derived cardiomyocytes treated for 24 hours with indicated chimeras, and (H) mouse hepatocytes (AML12 cell line) treated with pCox4-mCOX1^1-24 targeting mouse COX1 mRNA. Downregulated newly synthesized proteins are indicated by red dot. ATP6 signal reduction, upon ATP8 downregulation, is indicated with a gray dot.

Fig. 3. Silencing of mtDNA-encoded genes in living cells alters RNA abundance.

See also fig. S3, S4, and S5. See also table S1 and S2. (A) Heatmap representing the abundance of mtDNA-encoded mRNAs and rRNAs upon treatment with pCox4-COX1^1-19 compared to control (pCox4) as determined by nanoString. FC, fold change (mean, n=4). Statistical significance was determined by multiple t test using the Holm-Sidak method, with alpha=0.05 (^*, padj<0.05; ^**, padj<0.01; ^***, padj<0.001). (B) Effect of indicated chimera treatment on cognate mRNA abundance (mean ± SEM, n=3), compared to control (pCox4)(dashed line). (C) Mitochondrial translation silenced in cells for 48 hours for transcriptomics’ sample preparation. Newly-synthesized proteins were [³⁵S]methionine labelled. Targeted protein indicated by red dot; ATP6 upon ATP8 silencing indicated by gray dot. (D) Heatmap of clustering for significant variation of mRNA abundances upon 48 hours silencing. 1503 genes with significantly changed (padj<0.05) mRNA abundance under at least one condition (as in E) are shown. Total RNA was extracted from cells, mRNA enriched, and analyzed by RNA-seq. (E) Number of genes significantly changed (padj<0.05) upon treatment for each condition compared to the control (pCox4). The amount of downregulated (blue), upregulated (red), and total genes (at the bottom) are indicated for each gene silenced. (F) Data of altered mRNA abundance through UpSet visualization. Number of genes in each exclusive intersection is indicated for each subset as intersection size. The set size (same as total genes in E) is also represented. (G) Total genes significantly changed as represented in (E) were first grouped according to the related OXPHOS complexes into ND (ND1∪2∪3∪4L∪5), CYTB, COX (COX1∪2∪3), and ATP (ATP6∪8), and then represented by the UpSet algorithm. The size of each exclusive intersection is indicated, as well as the set size. (H) Gene functional enrichment analysis by gene ontology annotation of mRNAs that significantly varied in all groups (ND_CYTB_COX_ATP intersection) (I) Gene functional enrichment analysis by gene ontology annotation of mRNAs that significantly varied only upon silencing of complex IV subunits (COX).

Fig. 4. Proteome remodeling of mitochondria upon silencing in living cells.

See also fig. S6. See also table S3 and S4. (A-D) Mitochondrial OXPHOS complex activity after ND2, CYTB, COX1, and ATP8/ATP6 mRNA silencing in HEK293T cells for 48 hours. Represented as percentage of the control (n=4, mean ± SEM). Statistical significance was determined by unpaired t test (^**, p<0.01 and ^****, p<0.0001). (E) Immunodetection of mitochondrial OXPHOS complexes analyzed by BN-PAGE after treatment of HEK293T cells with chimeras silencing ND2, CYTB, COX1, and ATP8 for 48 hours. (F-G) Proteomic analyses of mitochondria isolated from cells treated with ND2^12-29 (F) and CYTB^6-24 (G) chimeras for 48 hours (n=4). Rank sum plots display variation in protein abundance between silenced cells (ND2^12-29 and CYTB^6-24), represented as fold change compared to the control. Proteins associated to assembly of complexes I, III, and IV indicated in green, orange, and blue, respectively. (H) Immunodetection of OXPHOS mitochondrial complexes by BN-PAGE after treatment of HEK293T cells with COX1^1-19 for 72 hours. CI, II, III, IV, & V: complex I, II, III, IV, & V, respectively. (I-K) Real time respirometry of HEK293T cells treated with pCox4-COX1^1-19 for 72 hours. The basal (J) and maximal (K) oxygen consumption rates (OCR) were significantly decreased compared to the control treatment (pCox4) (^****, p<0.0001 and ^**, p<0.01) (n=3, mean ± SEM). (L) Extracellular acidification rate (ECAR) in cells treated with pCox4-COX1^1-19 for 72 hours compared to the control (pCox4)(^*,p<0.05) (n=3, mean±SEM). (M) Mitochondrial membrane potential measured by TMRM in HEK293T cells treated with pCox4-COX1^1-19 for 72 hours (ns, not significant) (n=4, mean ± SEM). Statistical significance in (J-M) was determined by unpaired t test. (N) Effect of COX1 knockdown on the mitochondrial proteome analyzed by quantitative MS analyses of mitochondria from HEK293T cells treated with COX1^1-19 for 8, 16, 24, 48, and 72 hours. (O) Heatmap of quantitative MS analyses of complex IV subunits. (P) Diagram illustrating gradual and sequential propagation of OXPHOS defects upon COX1 silencing.

Fig. 5. Identification of mitochondrial biogenesis factors.

See also fig. S7. See also table S5. (A-C) Real time respirometry measurements of HEK293T after siRNA-mediated downregulation of TMEM186 (siTMEM186), ZNF703 (siZNF703), and LINC00493 (siLINC). Control, cells treated with non-targeting siRNA (siNT). Representative profile of oxygen consumption rate (OCR) (A), basal OCR (B) (mean ± SEM; n=5 for all except siLINC where n=3), and maximal OCR (C) (mean ± SEM; n=5 for all except siLINC where n=3). Statistical significance was determined by 2-way ANOVA with multiple comparisons (ns, not significant; ^*, p<0.05; ^**, p<0.01; ^****, p<0.0001). (D) Quantitative mass spectrometry analyses to determine TMEM186, ZNF703, and SMIM26 interaction partners. TMEM186^FLAG, ZNF703^FLAG, and SMIM26^FLAG were expressed in HEK293T cells, mitochondria isolated, solubilized, and subjected to FLAG-IP. Eluates (including the control) were analyzed by label-free mass spectrometry and protein enrichment represented as log₂ (Fold Change) (n=4). Significantly enriched proteins are indicated in orange. The bait is presented in bold with a star in each case. (E) TMEM186^FLAG, ZNF703^FLAG, and SMIM26^FLAG co-precipitated newly synthesized mtDNA-encoded proteins. Total, 3%; eluate 100%. (F) Determination of mitochondrial mRNAs and rRNAs co-precipitating with TMEM186^FLAG and SMIM26^FLAG by nanoString technology. Results are presented as fold change to the Control (WT cells, dashed line) (mean ± SEM, n=3). Statistical significance was determined by multiple t test using the Holm-Sidak method, with alpha=0.05 (^*, padj<0.05; ^**, padj<0.01; ^***, padj<0.001). (G) Mitochondrial translation products were labeled with [³⁵S]methionine after siRNA-mediated downregulation of LINC00493 (siLINC) in HEK293T cells. Control, non-targeting siRNA (siNT). (H-I) Immunodetection of mitochondrial respiratory complexes by BN-PAGE after siRNA-mediated downregulation of LINC00493 (siLINC) in HEK293T cells. Isolated mitochondria were solubilized with 1% DDM (H) or Digitonin (I). CI, complex I; CII, complex II; CIII, complex III; CIV, complex IV; CV, complex V.