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Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number.

Spadafora D, Kozhukhar N, Alexeyev MF - PLoS ONE (2016)

Bottom Line: Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content.Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number.This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of South Alabama, 307 University Blvd, Mobile, Alabama, 36688, United States of America.

ABSTRACT
Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.

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A method for establishing mouse cell lines with reduced mtDNA copy number.A and B, initial screening for mtDNA copy number in 3T3#53 clones expressing LigA, in which Lig3 exon 1 or exon 8 was targeted with CRISPR-Cas9. C and D, The relationship between Lig3 inactivation and mtDNA copy number in clones with targeted exon 1 and exon 8.
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pone.0152705.g007: A method for establishing mouse cell lines with reduced mtDNA copy number.A and B, initial screening for mtDNA copy number in 3T3#53 clones expressing LigA, in which Lig3 exon 1 or exon 8 was targeted with CRISPR-Cas9. C and D, The relationship between Lig3 inactivation and mtDNA copy number in clones with targeted exon 1 and exon 8.

Mentions: The procedure for the establishment of mouse cell lines with reduced mtDNA copy number described above relies on availability of cell lines with floxed Lig3 alleles, and therefore is difficult to generalize. To develop a general method, we designed four single guide RNAs (sgRNAs) for CRISPR-Cas9 mediated inactivation of the Lig3 gene in mouse cells by introducing deletions in exon 1 or in exon 8 of this gene (two sgRNAs for each exon, S1H Fig and S1 Table). The method was validated by inactivating Lig3 in 3T3#52 cells expressing LigA as described in the Materials and Methods. Initial screening of the clones with putative inactivation of the Lig3 revealed substantial heterogeneity in mtDNA copy number in clones in which exon 1 was targeted (Fig 7A). Interestingly, clones which retained Lig3 expression (possibly due to incomplete inactivation of all Lig3 alleles) had high mtDNA content (Fig 7C and 7D), whereas all tested clones with low mtDNA copy number had all Lig3 alleles inactivated (S3B Fig). Of note, clone B6 (Fig 7C), which had high mtDNA content, but no Lig3 detectable by western blotting, had one apparently active allele with two in-frame deletions (S3A Fig). High mtDNA copy number in this clone is consistent with our previous observation that trace amounts of Lig3 are sufficient for the maintenance of normal mtDNA copy number [19]. Remarkably, all tested clones in which exon 8 was targeted had reduced mtDNA copy number (Fig 7B, 7D and S3C Fig). This observation may reflect the fact that exon 8 contains the active site of the enzyme, and therefore both in-frame and out-of-frame deletions in this exon are likely to be detrimental. The reduction in mtDNA copy number in 3T3#52 cells induced by substitution of the Lig3 with LigA was stable (S3D Fig), which underscores the utility of the approach.


Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number.

Spadafora D, Kozhukhar N, Alexeyev MF - PLoS ONE (2016)

A method for establishing mouse cell lines with reduced mtDNA copy number.A and B, initial screening for mtDNA copy number in 3T3#53 clones expressing LigA, in which Lig3 exon 1 or exon 8 was targeted with CRISPR-Cas9. C and D, The relationship between Lig3 inactivation and mtDNA copy number in clones with targeted exon 1 and exon 8.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816344&req=5

pone.0152705.g007: A method for establishing mouse cell lines with reduced mtDNA copy number.A and B, initial screening for mtDNA copy number in 3T3#53 clones expressing LigA, in which Lig3 exon 1 or exon 8 was targeted with CRISPR-Cas9. C and D, The relationship between Lig3 inactivation and mtDNA copy number in clones with targeted exon 1 and exon 8.
Mentions: The procedure for the establishment of mouse cell lines with reduced mtDNA copy number described above relies on availability of cell lines with floxed Lig3 alleles, and therefore is difficult to generalize. To develop a general method, we designed four single guide RNAs (sgRNAs) for CRISPR-Cas9 mediated inactivation of the Lig3 gene in mouse cells by introducing deletions in exon 1 or in exon 8 of this gene (two sgRNAs for each exon, S1H Fig and S1 Table). The method was validated by inactivating Lig3 in 3T3#52 cells expressing LigA as described in the Materials and Methods. Initial screening of the clones with putative inactivation of the Lig3 revealed substantial heterogeneity in mtDNA copy number in clones in which exon 1 was targeted (Fig 7A). Interestingly, clones which retained Lig3 expression (possibly due to incomplete inactivation of all Lig3 alleles) had high mtDNA content (Fig 7C and 7D), whereas all tested clones with low mtDNA copy number had all Lig3 alleles inactivated (S3B Fig). Of note, clone B6 (Fig 7C), which had high mtDNA content, but no Lig3 detectable by western blotting, had one apparently active allele with two in-frame deletions (S3A Fig). High mtDNA copy number in this clone is consistent with our previous observation that trace amounts of Lig3 are sufficient for the maintenance of normal mtDNA copy number [19]. Remarkably, all tested clones in which exon 8 was targeted had reduced mtDNA copy number (Fig 7B, 7D and S3C Fig). This observation may reflect the fact that exon 8 contains the active site of the enzyme, and therefore both in-frame and out-of-frame deletions in this exon are likely to be detrimental. The reduction in mtDNA copy number in 3T3#52 cells induced by substitution of the Lig3 with LigA was stable (S3D Fig), which underscores the utility of the approach.

Bottom Line: Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content.Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number.This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of South Alabama, 307 University Blvd, Mobile, Alabama, 36688, United States of America.

ABSTRACT
Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.

Show MeSH
Related in: MedlinePlus