The long non-coding RNA Cerox1 is a post transcriptional regulator of mitochondrial complex I catalytic activity
- University of Edinburgh, Western General Hospital, United Kingdom
- University of Oxford, United Kingdom
- University of Edinburgh, United Kingdom
Figures
Figure 1 with 1 supplement
Cerox1 is an evolutionarily conserved, highly expressed and predominantly cytoplasmic lncRNA.
(A) The mouse Cerox1 locus (mm9 assembly). Sequence shaded in blue highlights conservation within exon two among eutherian mammals, but not in non-mammalian vertebrates such as chicken and …
Figure 1—figure supplement 1
Transcript characterisation.
(A) PhyloCSF tracks for mouse Cerox1 and Sox8 and human CEROX1 and SOX8. (B) Expression of Cerox1 (i) and Sox8 (ii) in the adult mouse subventricular zone. Cerox1 expression is particularly …
Figure 2 with 1 supplement
Cerox1 overexpression elevates levels of OXPHOS transcripts and their encoded proteins.
(A) Gene ontology analysis indicates a significant enrichment of upregulated genes involved in mitochondrial electron transport, energy production and redox reactions. (B) Four membrane bound …
-
Figure 2—source data 1
N2A metabolomics profiling - Figure 2E.
- https://doi.org/10.7554/eLife.45051.007
Figure 2—figure supplement 1
Manipulation of Cerox1 expression levels.
(A) Fold-change in Cerox1 level following treatment with each of twelve unique shRNAs. (B) CRISPR activation and inhibition at the divergent Cerox1-Sox8 promoter. (C) Overexpression and …
Figure 3 with 1 supplement
OXPHOS enzyme activity and oxygen consumption change concordantly and substantially with Cerox1 level alteration.
(A) Enzyme activities in mouse N2A cells 72 hr post-transfection of Cerox1 overexpression construct. Mouse Cerox1 overexpression in N2A cells results in significant increases in the catalytic …
-
Figure 3—source data 1
N2A Cerox1 overexpression specific enzyme assays - Figure 3A.
- https://doi.org/10.7554/eLife.45051.010
-
Figure 3—source data 2
N2A Cerox1 overexpression seahorse bioanalyzer - Figure 3B.
- https://doi.org/10.7554/eLife.45051.011
-
Figure 3—source data 3
N2A Cerox1 knock down specific enzyme assays - Figure 3C.
- https://doi.org/10.7554/eLife.45051.012
-
Figure 3—source data 4
N2A Cerox1 knock down seahorse bioanalyzer - Figure 3D.
- https://doi.org/10.7554/eLife.45051.013
Figure 3—figure supplement 1
Increases in mitochondrial complex I and complex IV activities are not due to an increase in mitochondrial copy number.
(A) The mitochondrial-to-nuclear genome ratio was calculated by amplifying the nuclear genes β-actin and beclin-1 and the mitochondrial genes Nd1 and Nd2 and calculating the ratio. Primers were …
Figure 4
Cellular oxidative stress and viability depend on Cerox1 levels.
(A) Cerox1 knockdown increases the production of reactive oxygen species by 20%, whilst Cerox1 overexpression decreases it by 45% (error bars s.e.m., n = 12 biological replicates). (B) Protein …
-
Figure 4—source data 1
N2A Reactive oxygen species production - Figure 4A.
- https://doi.org/10.7554/eLife.45051.015
-
Figure 4—source data 2
N2A Cerox1 overexpression protein carbonylation - Figure 4B.
- https://doi.org/10.7554/eLife.45051.016
-
Figure 4—source data 3
N2A cell viability Cerox1 overexpression and knockdown - Figure 4C.
- https://doi.org/10.7554/eLife.45051.017
Figure 5 with 1 supplement
The effect of Cerox1 on complex I transcript levels is miRNA-dependent.
(A) Overexpression of Cerox1 in mouse wildtype and DicerΔ/Δembryonic stem (ES) cells (inset graph). The overexpression of Cerox1 in wildtype mouse embryonic stem cells results in an increase in …
-
Figure 5—source data 1
N2A wildtype and MRE mutant Cerox1 overexpression specific enzyme assays - Figure 5D.
- https://doi.org/10.7554/eLife.45051.020
Figure 5—figure supplement 1
Expression profile of mmu-miR-488-3p.
(A) Expression profile of mouse miR-28–5p, miR-138–5p, miR-370–3p, miR-488–3p and miR-708–5p in N2A cells as measured by quantitative PCR and nanostring. Both techniques reveal the same trend in …
Figure 6
An intact miR-488–3p response element site is required for the effect of Cerox1 on complex I catalytic activity.
(A) Overexpression of miR-488–3p knocks down all Cerox1-sensitive subunit transcripts. Error bars s.e.m. (n = 3 biological replicates for control and overexpression of miR-488–3p). 2-sided t-test; …
-
Figure 6—source data 1
N2A wildtype and miR-488–3 p mutant Cerox1 overexpression complex I and citrate synthase assays - Figure 6F.
- https://doi.org/10.7554/eLife.45051.022
Figure 7
Human CEROX1 modulates complex I activity in mouse cells.
(A) CEROX1 is enriched in the cytoplasm. Error bars s.e.m. n = 4 biological replicates. (B) Relative levels of lncRNA (blue) and protein-coding gene (grey) expression across individuals and tissues …
-
Figure 7—source data 1
HEK293T CEROX1 overexpression specific enzyme assays - Figure 7D.
- https://doi.org/10.7554/eLife.45051.024
-
Figure 7—source data 2
HEK293T CEROX1 overexpression seahorse bioanalyzer - Figure 7E.
- https://doi.org/10.7554/eLife.45051.025
-
Figure 7—source data 3
Reciprocal overexpression, complex I and citrate synthase assay - Figure 7F.
- https://doi.org/10.7554/eLife.45051.026
Figure 8
Proposed model for Cerox1 as a post-transcriptional regulator of mitochondrial protein production and energy metabolism.
In this model, Cerox1 (A) post-transcriptionally maintains energy metabolism homeostasis through buffering the stable ETC transcripts against miRNA-mediated gene silencing. Overexpression of Cerox1 …
Author response image 1
Author response image 2
Author response image 3
Author response image 4
Author response image 5
Author response image 6
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | Cerox1 | NA | AK079380; 2810468N07Rik; ENSMUST00000163493 | |
| Gene (Homo sapiens) | CEROX1 | NA | BC098409; RP11-161M6.2; ENST00000562570 | |
| Cell line (Mus musculus) | N2A (mouse neuroblastoma cells) | European Collection of Authenicated Cell Cultures (ECACC) | RRID: CVCL_0470; ECACC: 89121404 | |
| Cell line (Homo sapiens) | HEK293T (human embryonic kidney cells) | European Collection of Authenicated Cell Cultures (ECACC) | RRID: CVCL_0063; ECACC 12022001 | |
| Transfected construct (Mus musculus and Homo sapiens) | pCAG-GFP | https://www.addgene.org/89684/ | RRID:Addgene_89684 | This backbone was modified for overexpression of Cerox1, Cerox1 MRE mutants and CEROX1 |
| Transfected construct (Mus musculus) | BLOCK-it U6 shRNA expression construct | Invitrogen | K494500 | |
| Transfected construct (Mus musculus) | BLOCK-iT Pol II miR RNAi expression vector | Invitrogen | K493600 | |
| Antibody | anti-NDUFS1, rabbit monoclonal | Abcam | RRID: AB_2687932; ab169540 | Overnight, four degrees (1:30,000) |
| anti-NDUFS3, mouse monoclonal | Abcam | RRID:AB_10861972; ab110246 | Overnight, four degrees, 0.15 mg/ml | |
| anti-alpha tubulin, mouse monoclonal | Abcam | RRID:AB_2241126; ab7291 | Overnight, four degrees (1:30,000) | |
| goat anti-rabbit HRP, goat polyclonal | Invitrogen | RRID:AB_2536530; G-21234 | Room temperature,1 hr (1:30,000) | |
| goat anti-mouse HRP, goat polyclonal | Dako | RRID:AB_2617137; P0447 | Room temperature,1 hr (1:3,000) | |
| Recombinant DNA reagent | Fugene6 | Promega | E2691 | |
| Commercial assay or kit | Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit | Thermofisher | A22188 | |
| OxyBlot protein oxidation detetion kit | Merck Millipor | S7150 | ||
| QuantiGene ViewRNA miRNA ISH cell assay kit | Affymetrix | QVCM0001 | ||
| Chemical compound, drug | Rotenone | Sigma Aldrich | R8875-5G | |
| Antimycin A | Sigma Aldrich | A8674-25MG | ||
| Sodium Azide | Sigma Aldrich | S8032-25G | ||
| Oligomycin | Sigma Aldrich | 75351–5 MG | ||
| NADH (reduced) | Sigma Aldrich | N8129-1G | ||
| coenzyme Q | Sigma Aldrich | C7956-2mg | ||
| Fatty acid free albumin | Sigma Aldrich | A8806-1G | ||
| Sodium succinate | Sigma Aldrich | S5047-100G | ||
| Dichlorophenolindophenol (DCPIP) | Sigma Aldrich | 33125–5 G-R | ||
| Decylubiquinone | Sigma Aldrich | D7911-10mg | ||
| cytochrome c | Sigma Aldrich | C7752 | ||
| Carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone | Sigma Aldrich | C2920-10mg | ||
| miRNA inhibitors | Ambion | 4464084 | ||
| miRCURY LNA biotinylated miRNAs | Exiqon | 339178 | ||
| Software, algorithm | TargetScan v7.0 | Agarwal et al., 2015 | DOI: 10.7554/eLife.05005 |
Additional files
-
Supplementary file 1
Association of CEROX1 single nucleotide polymorphism on anthropomorphic traits.
Data was accessed through http://geneatlas.roslin.ed.ac.uk/.
- https://doi.org/10.7554/eLife.45051.028
-
Supplementary file 2
Differentially expressed genes after overexpression of mouse Cerox1.
- https://doi.org/10.7554/eLife.45051.029
-
Supplementary file 3
MRE predictions for Nduf subunit transcripts.
- https://doi.org/10.7554/eLife.45051.030
-
Supplementary file 4
shRNA oligos and PCR primers.
- https://doi.org/10.7554/eLife.45051.031
-
Transparent reporting form
- https://doi.org/10.7554/eLife.45051.032
