Codon optimization is an essential parameter for the efficient allotopic expression of mtDNA genes

It may state two separate claims as one.

Eight of thirteen mitochondria-encoded oxidative phosphorylation (OxPhos) proteins maintained protein expression following stable selection, with mitochondrial localization of expression products.

“Eight of thirteen mitochondria-encoded oxidative phosphorylation (OxPhos) proteins maintained protein expression following stable selection, with mitochondrial localization of expression products.”

It may state two separate claims as one.

Of all thirteen minimally recoded genes, only rND4 demonstrated any stable protein expression, and this was only a fraction of that detected for oND4 under similar conditions.

“Of all thirteen minimally recoded genes, only rND4 demonstrated any protein expression stably; however, this was only a fraction of that detected for oND4 under similar conditions.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

We have developed a protocol to enrich mitochondria from limited numbers of mammalian cells.

“We have developed a protocol to rapidly and economically enrich mitochondria from limited numbers of mammalian cells.”

It may state two separate claims as one.

Severe mutations in mitochondrial subunits, such as the ND1m mutant line, disrupt OXPHOS to a degree that proliferation rates are markedly reduced even under normal growth conditions, with dramatically impaired viability in glucose-restricted media demanding oxidative metabolism.

“Severe mutations in mitochondrial subunits, such that of the ND1m mutant line, disrupt OXPHOS to a degree that proliferation rates are markedly reduced even under normal growth conditions, with dramatically impaired viability in glucose-restricted media demanding oxidative metabolism.”

It may state two separate claims as one.

When cultured in glucose-containing media, ND1m cells proliferated at approximately 25% the rate of wild-type 143B cells, and growth rate of ND1m cells expressing a tagged oND1 was not significantly increased from the mutant.

“when cultured in glucose-containing media, ND1m cells appeared to proliferate at approximately 25% the rate of wild-type 143B cells, and growth rate of ND1m cells expressing a tagged oND1 was not significantly increased from the mutant.”

It may state two separate claims as one.

In partially reverted ND1m/+ lines, the FLAG epitope incorporates into both Complex I monomers and supramolecular structures, comigrating with endogenous ND1 subunit.

“In partially reverted ND1m/+ lines, the FLAG epitope clearly incorporates into both Complex I monomers and supramolecular structures, comigrating with the endogenous ND1 subunit”

It may state two separate claims as one.

Codon-optimized oND1 can incorporate and partially restore OxPhos function in an ND1 null background, with greatly enhanced incorporation under heteroplasmic ND1m/+ conditions.

“We also show that codon optimized oND1 can incorporate and partially restore OxPhos function in an ND1 null background, with greatly enhanced incorporation under heteroplasmic ND1m/+ conditions.”

Probably not a substantial claim.

The ND1m cell line demonstrates markedly slower growth under glucose conditions relative to wild-type.

“Because the ND1m line demonstrates markedly slower growth under glucose conditions relative to wild-type”

It may state two separate claims as one.

The majority of mRNAs from stably-selected HEK293 cells are equal or one to two orders of magnitude lower in expression than GAPDH mRNA, and the most robustly expressed optimized gene (oND3) is present at no more than 1.38 times GAPDH.

“the majority of mRNAs are equal or one to two orders of magnitude lower in expression than the mRNA of housekeeping gene GAPDH (Fig. 3B), and the most robustly expressed optimized genes are present at no more than 1.38 times GAPDH (for oND3)”

It may state two separate claims as one.

Complex I-associated Complex III was only present in WT mitochondria and those expressing oND1, confirming failed CI/III association in the ND1 null line and supporting some restored complex assembly upon allotopic expression of codon-optimized ND1 subunit.

“Complex I-associated Complex III was only present in WT mitochondria and those expressing oND1, as evidenced by the comigration of CIII with the GRIM-19 and NDUFA9 subunits of CI, thus confirming failed CI/III association in the ND1 null line and supporting some restored complex assembly upon allotopic expression of the codon-optimized ND1 subunit”

It may state two separate claims as one.

Under DDM conditions, monomeric CI is observed exclusively in wild-type 143B mitochondria and in ND1m mitochondria with allotopically-expressed oND1-FLAG, and is entirely absent in the ND1 null line.

“Under these conditions, monomeric CI is observed exclusively in wild-type 143B mitochondria and in ND1m mitochondria with allotopically-expressed oND1-FLAG, and is entirely absent in the ND1 null line.”

It may state two separate claims as one.

Of the minimally-recoded versions, only rND4 had any appreciable protein expression in stably selected HEK293 cells, though at levels well below its codon-optimized counterpart.

“of the minimally-recoded versions for each gene, only rND4 had any appreciable protein expression, though with detectable levels still well below its codon-optimized counterpart”

It may state two separate claims as one.

All recoded genes demonstrate strongly biased use of underrepresented codons UUA or CUA, and none use the dominant CUG codon.

“none use the dominant CUG codon, and all recoded genes instead demonstrate strongly biased use of underrepresented codons UUA or CUA.”

It may state two separate claims as one.

Viability of wild type 143B cells was reduced to ~55% when cultured in OxPhos restrictive media, and ND1m cells were only ~11% viable.

“the viability of wild type 143B cells was reduced to ~55% its viability when cultured in OxPhos restrictive media, and ND1m cells were only ~11% viable.”

It may state two separate claims as one.

Stably selected ND1m cells coding for oND1 expressed the FLAG epitope on denaturing western blots of mitochondrial protein isolates, with a major product at ~37 kDa and a minor product near 60 kDa, corresponding to processed and unprocessed ND1 products.

“Stably selected ND1m cells coding for oND1 expressed the FLAG epitope on denaturing western blots of mitochondrial protein isolates (Fig. 4C), with a major product detected at ~ 37 kDa and a minor product near 60 kDa, corresponding to the processed and unprocessed ND1 products, respectively.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Leucine constitutes 17% of the residues in mitochondrial protein-coding genes.

“which alone constitutes 17% of the residues in mitochondrial protein-coding genes.”

It may state two separate claims as one.

CI-linked activity in-gel was present in CI monomers in wild-type and oND1-expressing mitochondria, with no detectable CI function in the ND1 mutant cell line, suggesting that incorporated FLAG-tagged ND1 protein can support functional Complex I activity.

“Activity in wild-type and oND1-expressing mitochondria was present in CI monomers when lysed with DDM, with no detectable CI function in the ND1 mutant cell line (Fig. 4E), suggesting that the incorporated FLAG-tagged ND1 protein can indeed support functional Complex I activity.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

A no-template control was included in each reaction set to rule out non-specific priming.

“A no-template control was included in each reaction set to rule out non-specific priming.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Homoplasmy in the mouse cell lines was ascertained by Sanger sequencing.

“Homoplasmy in the mouse cell lines C57/BL6(WT), C57/BL6(mtFVB) and C57/BL6(mtFVB) stably expressing doxycycline inducible allotopic MsoATP8 was ascertained by Sanger sequencing using the primer indicated in Fig. S3B.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The C57/BL6(mtFVB) mutation has been shown to cause various defects in inbred mice including anxiety and inflammation-related arthritis.

“The C57/BL6(mtFVB) mutation has been shown to cause various defects in inbred mice including anxiety and inflammation-related arthritis [91,92].”

• cites [91] Weiss, 2012, The mitochondrial Atp8 mutation induces mitochondrial ROS generation, secretory dysfunction, and beta-cell mass adaptation in conplastic B6-mtFVB mice, Endocrinology sourceNot checked — no full text of the source
• cites [92] Yu, 2009, The mtDNA nt7778 G/T polymorphism affects autoimmune diseases and reproductive performance in the mouse, Hum. Mol. Genet. sourceNot checked — no full text of the source

It may state two separate claims as one.

Optimizing the primary sequence of mitochondrial genes facilitates their nuclear expression in human cells and, in some cases, their assembly into respiratory complexes.

“Here, we have demonstrated that merely optimizing the primary sequence of mitochondrial genes facilitates their nuclear expression in human cells, and in some cases, their assembly into respiratory complexes”

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

Mitochondrial-nuclear crosstalk is well documented, and evidence suggests that mutations in the mitochondrial genome influence nuclear gene expression profiles even when phenotypes are mild.

“mitochondrial-nuclear crosstalk is well documented, and evidence suggests that mutations in the mitochondrial genome also influence nuclear gene expression profiles, even when phenotypes are mild [94].”

• cites [94] Latorre-Pellicer, 2016, Mitochondrial and nuclear DNA matching shapes metabolism and healthy ageing, ON Nat. sourceNot checked — no full text of the source

It may state two separate claims as one.

The mutation m.3751insC in MT-ND1 introduces an additional C into a homopolymeric cytosine stretch, resulting in a premature stop codon (p.Leu89Profs*13) and truncation of the 318 amino acid protein product to 100 residues.

“This mutation introduces an additional C into a homopolymeric cytosine stretch in the first third of MT-ND1 (m.3751insC), resulting in a premature stop codon (p.Leu89Profs*13) and truncation of the 318 amino acid protein product to 100 residues”

• cites [73] Lim, 2016, Loss of mitochondrial DNA-encoded protein ND1 results in disruption of complex I biogenesis during early stages of assembly, FASEB J. sourceNot checked — no full text of the source
• cites [79] McKenzie, 2014, Capture of somatic mtDNA point mutations with severe effects on oxidative phosphorylation in synaptosome cybrid clones from human brain, Hum. Mutat. sourceCheck pending

Probably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds.

The method is comparable to commercially available kits.

“and is comparable to commercially available kits”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

ARMS qPCR was used to ascertain mutation load in cybrids during the study.

“We used Amplification Refractory Mutation System-quantitative PCR (ARMS qPCR) [95] to ascertain mutation load in cybrids during the course of this study.”

• cites [95] Bai, 2004, Detection and quantification of heteroplasmic mutant mitochondrial DNA by real-time amplification refractory mutation system quantitative PCR analysis: a single-step approach, Clin. Chem. sourceNot checked — no full text of the source

Probably not a substantial claim. The paper may give no reason why it holds.

A critical but often-overlooked consideration in nuclear relocation studies is the influence of the primary coding sequence on protein production.

“A critical, but often-overlooked consideration in these nuclear relocation studies, however, is the influence of the primary coding sequence on protein production.”

It may state two separate claims as one.

ND1 has previously been allotopically expressed to demonstrate the necessity of functional CI in the hypoxia-induced shift to a Warburg profile during tumorigenesis, but this was also only achieved under conditions of heteroplasmy.

“ND1 has previously been allotopically expressed to demonstrate the necessity of functional CI in the hypoxia-induced shift to a Warburg profile during tumorigenesis, however this was also only achieved under conditions of heteroplasmy [30].”

• cites [30] Nagley, 1989, Leading organellar proteins along new pathways: the relocation of mitochondrial and chloroplast genes to the nucleus, Trends Biochem. Sci. sourceNot checked — no full text of the source

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

The protease cleavage site of the ATP5G1 MTS has been determined, and the first five amino acids of mature ATP5G1 protein were included to facilitate processing.

“The protease cleavage site of this MTS has been determined and, for this study, we also included the first five amino acids of the mature ATP5G1 protein to facilitate processing of the precursor [36].”

• cites [36] Manfredi, 2002, Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene, to the nucleus, Nat. Genet. sourceNot checked — no full text of the source

The quote only partly states this claim.

Homoplasmy was preserved throughout the duration of lengthy experiments, as tested by ARMS qPCR at various time points.

“in order to confirm that homoplasmy was preserved throughout the duration of lengthy experiments, we tested the mutation load in cybrid cell lines at various time points by ARMS qPCR.”

It may state two separate claims as one.

Codon-optimized genes are comprised overwhelmingly of codons with nuclear use bias and avoid rare codons, likely contributing to their enhanced nuclear expression over minimally recoded versions.

“codon-optimized genes are comprised overwhelmingly of codons which show use bias in nuclear genes and avoid the use of underrepresented, or rare codons, likely contributing to their enhanced nuclear expression over minimally recoded versions.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The method is suitable to process multiple samples in parallel.

“This method is suitable to process multiple samples in parallel”

It may state two separate claims as one.

In the absence of wild-type ND1, Complex I fails to assemble, resulting in impaired oxidative metabolism and failure to grow in galactose media.

“In the absence of wild-type ND1, Complex I fails to assemble, resulting in impaired oxidative metabolism and failure to grow in galactose media [34,80].”

• cites [34] Iommarini, 2018, Unravelling the effects of the mutation m.3571insC/MT-ND1 on respiratory complexes structural organization, Int. J. Mol. Sci. sourceCheck pending
• cites [80] Antonicka, 2003, Identification and characterization of a common set of complex I assembly intermediates in mitochondria from patients with complex I deficiency, J. Biol. Chem. sourceCheck pending

It may state two separate claims as one.

The authors previously reported successful allotopic expression of MT-ATP8 and, to a lesser extent, MT-ATP6, with functional rescue in the A8/A6mut cybrid null for ATP8 protein.

“We previously reported the successful allotopic expression of the MT-ATP8 gene and to a lesser extent the MT-ATP6 gene, with functional rescue in a cybrid cell line A8/A6mut (m.8529G-A) null (Fig. 5A) for the ATP8 protein [87].”

• cites [87] Boominathan, 2016, Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant, Nucleic Acids Res. sourceNot checked yet

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

MT-ATP8 and MT-ATP6 are mtDNA-encoded subunits of ATP synthase, while the remaining subunits are nuclear-encoded.

“Two of the subunits of this multimeric complex are encoded by mtDNA, namely MT-ATP8 and MT-ATP6, whereas, the genes for the remaining subunits reside in the nucleus [84].”

• cites [84] Jonckheere, 2012, Mitochondrial ATP synthase: architecture, function and pathology, J. Inherit. Metab. Dis. sourceCheck pending

It may state two separate claims as one.

C57/BL6(mtFVB) mice demonstrate measurable phenotypes and their cells in vitro have subtle deficits indicative of disrupted Complex V function.

“C57/BL6(mtFVB) mice demonstrate measurable phenotypes and cells in vitro have subtle deficits indicative of disrupted CV function [90,91]”

• cites [90] Eipel, 2011, Mutation of mitochondrial ATP8 gene improves hepatic energy status in a murine model of acute endotoxemic liver failure, Life Sci. sourceNot checked — no full text of the source
• cites [91] Weiss, 2012, The mitochondrial Atp8 mutation induces mitochondrial ROS generation, secretory dysfunction, and beta-cell mass adaptation in conplastic B6-mtFVB mice, Endocrinology sourceNot checked — no full text of the source

Probably not a substantial claim.

The A8/A6mut cells were selected for homoplasmy as described previously.

“The A8/A6mut cells were selected for homoplasmy as described previously [87].”

• cites [87] Boominathan, 2016, Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant, Nucleic Acids Res. sourceNot checked yet

It may state two separate claims as one.

A heteroplasmic model remains more representative of dysfunctional mitochondrial conditions in affected patients, and therefore incorporation of allotopically expressed subunits in this context remains a clinically relevant result.

“a heteroplasmic model remains more representative of dysfunctional mitochondrial conditions in affected patients, and therefore the incorporation of allotopically expressed subunits in this context remains a clinically relevant result.”

It may state two separate claims as one.

Minimally-recoded gene sequences largely reflect mitochondrial codon preferences, while codon-optimized genes should have coding patterns more similar to nuclear genes.

“Because minimally-recoded gene sequences have only the changes necessary to preserve the amino acid sequence of a protein, they largely reflect codon preferences of the mitochondrial genome, while genes codon-optimized for nuclear expression should demonstrate coding patterns more similar to those of nuclear genes.”

It may state two separate claims as one.

The high bias toward underrepresented nuclear codons for these amino acids reflects a preference for these codons in mitochondrial translation and suggests they may be poorly translated in the cytosolic context.

“the high bias toward underrepresented nuclear codons for these amino acids reflects a preference for these codons in mitochondrial translation, and meanwhile suggests they may be poorly translated in the cytosolic context.”

• AssumptionRests on an assumption: Synonymous codon use of a genome can be loosely associated with translation.

Probably not a substantial claim.

The vast majority of previous allotopic expression studies have utilized minimally-recoded mitochondrial genes, wherein only bases differing from the universal genetic code are changed.

“The vast majority of these previous studies have utilized what may be considered “minimally-recoded” mitochondrial genes, wherein the only bases changed are those which differ from the universal genetic code (Trp, Met, Stop)”

It may state two separate claims as one.

Mitochondrial genes utilize synonymous codons differently than nuclear genes, featuring a high percentage of codons rare or underrepresented in the nuclear genome, which may account for poor or absent allotopic expression of minimally-recoded genes.

“Analysis of codon use patterns in minimally-recoded and codon-optimized gene constructs revealed that mitochondrial genes indeed utilize synonymous codons differently than nuclear genes, featuring a high percentage of codons considered to be rare or underrepresented in the nuclear genome, which may account for the poor or absent allotopic expression of minimally-recoded genes.”

It may state two separate claims as one.

Elements including relative frequencies of codon use, local and global GC composition, mRNA secondary structure stability, and presence of cryptic termination signals or splice sites are significant factors impacting protein expression.

“elements including the relative frequencies of codon use for each amino acid, local and global GC composition, mRNA secondary structure stability, and the presence of cryptic termination signals or splice sites are significant factors impacting the degree of protein expression”

• cites [61] Hanson, 2018, Codon optimality, bias and usage in translation and mRNA decay, Nat. Rev. Mol. Cell Biol. sourceNot checked yet
• cites [62] Inouye, 2015, Codon optimization of genes for efficient protein expression in mammalian cells by selection of only preferred human codons, Protein Expr. Purif. sourceNot checked — no full text of the source
• cites [63] Stachyra, 2016, Codon optimization of antigen coding sequences improves the immune potential of DNA vaccines against avian influenza virus H5N1 in mice and chickens, Virol. J. sourceCheck pending

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The protocol rapidly and economically enriches mitochondria from limited numbers of mammalian cells.

“to rapidly and economically enrich mitochondria from limited numbers of mammalian cells”

It may state two separate claims as one.

The codon use profiles of mitochondrial genes are significant in facilitating heterologous nuclear expression and promoting effective translation.

“the codon use profiles of mitochondrial genes are significant in facilitating heterologous nuclear expression and promoting effective translation.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Pathological mutations in MT-ATP8 and MT-ATP6 manifest as NARP, MILS, and cardiac hypertrophy.

“Several pathological mutations in these two mtDNA subunits have been reported and manifest as NARP (Neuropathy, Ataxia, and Retinitis Pigmentosa), MILS (Maternally Inherited Leigh Syndrome) (www.mitomap.org), and cardiac hypertrophy [85,86] in patients.”

• cites [85] Jonckheere, 2008, A novel mitochondrial ATP8 gene mutation in a patient with apical hypertrophic cardiomyopathy and neuropathy, J. Med. Genet. sourceCheck pending
• cites [86] Ware, 2009, Infantile cardiomyopathy caused by a mutation in the overlapping region of mitochondrial ATPase 6 and 8 genes, J. Med. Genet. sourceNot checked — no full text of the source

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

The F1Fo ATP synthase is the terminal complex in oxidative phosphorylation and converts the proton motive force generated by Complexes I, III and IV into ATP.

“The F1Fo ATP synthase is the terminal complex in the oxidative phosphorylation relay and is responsible for converting the proton motive force generated by Complexes I, III and IV into ATP.”

• cites [84] Jonckheere, 2012, Mitochondrial ATP synthase: architecture, function and pathology, J. Inherit. Metab. Dis. sourceCheck pending

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Both inherited and acquired mutations in mtDNA cause severe diseases including LHON, Leigh's syndrome, MELAS, MERRF, NARP and MILS, Kern-Sayer's syndrome, and reversible respiratory chain syndrome.

“Both inherited and acquired mutations in mtDNA cause severe diseases, including LHON, Leigh’s syndrome, MELAS, MERRF, NARP and MILs, Kern-Sayer’s syndrome, and reversible respiratory chain syndrome (reviewed in 2).”

• cites [2] Chinnery, 2000, Mitochondrial DNA mutations in the pathogenesis of human disease, Mol. Med. Today sourceNot checked — no full text of the source

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

mtDNA mutations accumulate with age and accompany a progressive decline in organelle function.

“mtDNA mutations also accumulate with age and accompany a progressive decline in organelle function [4].”

• cites [4] Schaefer, 2008, Prevalence of mitochondrial DNA disease in adults, Ann. Neurol. sourceNot checked — no full text of the source

Probably not a substantial claim. The paper’s conclusions may not depend on it.

RSCU values were calculated to address inherent bias due to the codon composition of hydrophobic amino acids.

“therefore RSCU values were calculated to address inherent bias due to the codon composition of hydrophobic amino acids (Table S2).”

It may state two separate claims as one.

Although changes to coding sequence alone may not be sufficient for complete complementation of all mtDNA genes, host nuclear codon optimization is, for most, sufficient for stable expression.

“we conclude that although making changes to coding sequence alone may not be sufficient for complete complementation of all mtDNA genes, host (nuclear) codon optimization is, for most, sufficient for stable expression”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Clinical gene therapy using a codon-optimized exogenous construct to compensate for mutations in a nuclear gene is ongoing.

“clinical gene therapy using a codon-optimized exogenous construct to compensate for mutations in a nuclear gene (e.g. hemophilia, [69]) is ongoing”

• cites [69] Rangarajan, 2017, AAV5-Factor VIII gene transfer in severe hemophilia A, N. Engl. J. Med. sourceNot checked yet

It may state two separate claims as one.

The ND1m cells were previously shown to be homoplasmic and were confirmed to be >99.9% homoplasmic by ARMS qPCR and Sanger sequencing.

“The ND1m cells were previously shown to be homoplasmic [79] and within our experimental limitations we were able to confirm that these cells were >99.9% homoplasmic by ARMS qPCR (Supplementary Table S4) and Sanger sequencing (Supplementary Fig. S3).”

• cites [79] McKenzie, 2014, Capture of somatic mtDNA point mutations with severe effects on oxidative phosphorylation in synaptosome cybrid clones from human brain, Hum. Mutat. sourceCheck pending