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Overexpression of the mitochondrial methyltransferase TFB1M in the mouse does not impact mitoribosomal methylation status or hearing.

Lee S, Rose S, Metodiev MD, Becker L, Vernaleken A, Klopstock T, Gailus-Durner V, Fuchs H, Hrabě De Angelis M, Douthwaite S, Larsson NG - Hum. Mol. Genet. (2015)

Bottom Line: Non-syndromic deafness and predisposition to aminoglycoside-induced deafness can be caused by specific mutations in the 12S rRNA gene of mtDNA and are thus maternally inherited traits.In contrast, it was recently reported that signaling induced by 'hypermethylation' of two conserved adenosines of 12S rRNA in the mitoribosome is of key pathophysiological importance in sensorineural deafness.We thus conclude that therapies directed against mitoribosomal methylation are unlikely to be beneficial to patients with sensorineural hearing loss or other types of mitochondrial disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Karolinska Institutet, Retzius väg 8, 171 77 Stockholm, Sweden.

No MeSH data available.


Related in: MedlinePlus

Effect of TFB1M overexpression on mitochondria. (A) Quantitative RT–PCR analysis of mtDNA (CoxI) to nuclear DNA (beta-actin) ratios in hearts from wild-type controls (WT) and BAC-TFB1M (C7.1) transgenic mice at 20 weeks of age (n = 3 of each genotype). Error bars represent ±SD. (B) Steady-state levels of TFB1M protein in heart (upper panel) and liver (lower panel) from heterozygous TFB1M KO (+/−), wild-type (+/+) and heterozygous BAC-TFB1M transgenic mice (+/T) and the previously published mouse model overexpressing TFB1M (Tg-mtTFB1) as analyzed by immunoblotting of total protein extracts (30). Heart extracts from tissue-specific TFB1M KO mice (−/−) at 10 weeks of age were used as a control. Tubulin was used as a loading control. (C) Steady-state levels of OXPHOS subunits as determined by immunoblotting of total protein extracts from heart (left) or liver (right) from heterozygous TFB1M KO (+/−), wild-type (+/+) and heterozygous BAC-TFB1M transgenic mice (+/T) at 20 weeks of age. Heart extracts from tissue-specific TFB1M KO mice (−/−) at 10 weeks of age were used as a control. The following subunits were analyzed: Complex I (CI-NDUFB8), Complex II (CII-SDHB), Complex III (CIII-UQCRC2), Complex IV (CIV-MTCOI) and Complex V (CV-ATP5A).
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DDV427F3: Effect of TFB1M overexpression on mitochondria. (A) Quantitative RT–PCR analysis of mtDNA (CoxI) to nuclear DNA (beta-actin) ratios in hearts from wild-type controls (WT) and BAC-TFB1M (C7.1) transgenic mice at 20 weeks of age (n = 3 of each genotype). Error bars represent ±SD. (B) Steady-state levels of TFB1M protein in heart (upper panel) and liver (lower panel) from heterozygous TFB1M KO (+/−), wild-type (+/+) and heterozygous BAC-TFB1M transgenic mice (+/T) and the previously published mouse model overexpressing TFB1M (Tg-mtTFB1) as analyzed by immunoblotting of total protein extracts (30). Heart extracts from tissue-specific TFB1M KO mice (−/−) at 10 weeks of age were used as a control. Tubulin was used as a loading control. (C) Steady-state levels of OXPHOS subunits as determined by immunoblotting of total protein extracts from heart (left) or liver (right) from heterozygous TFB1M KO (+/−), wild-type (+/+) and heterozygous BAC-TFB1M transgenic mice (+/T) at 20 weeks of age. Heart extracts from tissue-specific TFB1M KO mice (−/−) at 10 weeks of age were used as a control. The following subunits were analyzed: Complex I (CI-NDUFB8), Complex II (CII-SDHB), Complex III (CIII-UQCRC2), Complex IV (CIV-MTCOI) and Complex V (CV-ATP5A).

Mentions: Mice that ubiquitously overexpressed TFB1M were created using a bacterial artificial chromosome (BAC) transgenic approach (32–34). We routinely use this approach as it is relatively insensitive to positional effects and results in low-to-moderate ubiquitous overexpression of nucleus-encoded mitochondrially targeted proteins (32–34). The recombinant Tfb1m gene (BAC-TFB1M) and its transcript could be tracked via a silent point mutation that was introduced to abolish an Xma I site in exon 3 (Fig. 2A). The BAC transgenic line C7.1 exhibited ubiquitous TFB1M overexpression levels (Fig. 2B–D) similar to those in the previously published mtTFB1 transgenic mouse line (30) (Fig. 3B) and was selected for further studies. To check for unwanted effects caused by genes flanking Tfb1m in the BAC clone, we generated transgenic mice harboring a version of the BAC clone (BAC-KO) in which Tfb1m expression was silenced by deletion of exon 3. Both the BAC-KO mice and the BAC transgenic C6.1 line, which only showed a minor increase of TFB1M mRNA expression (Fig. 2B), appeared to be completely healthy. A more marked increase in TFB1M expression was observed in the C7.1 line (Fig. 2B–D), and these transgenic mice were subjected to extensive phenotypic analyses. No changes were detected in their metabolism, cardiovascular function, lung function, eye function, grip strength or rotarod performance; similarly, hematological analyses of peripheral blood, broad spectrum clinical chemistry of plasma samples and pathology studies of various tissues showed no evidence of any change that could be related to the C7.1 genotype.Figure 2.


Overexpression of the mitochondrial methyltransferase TFB1M in the mouse does not impact mitoribosomal methylation status or hearing.

Lee S, Rose S, Metodiev MD, Becker L, Vernaleken A, Klopstock T, Gailus-Durner V, Fuchs H, Hrabě De Angelis M, Douthwaite S, Larsson NG - Hum. Mol. Genet. (2015)

Effect of TFB1M overexpression on mitochondria. (A) Quantitative RT–PCR analysis of mtDNA (CoxI) to nuclear DNA (beta-actin) ratios in hearts from wild-type controls (WT) and BAC-TFB1M (C7.1) transgenic mice at 20 weeks of age (n = 3 of each genotype). Error bars represent ±SD. (B) Steady-state levels of TFB1M protein in heart (upper panel) and liver (lower panel) from heterozygous TFB1M KO (+/−), wild-type (+/+) and heterozygous BAC-TFB1M transgenic mice (+/T) and the previously published mouse model overexpressing TFB1M (Tg-mtTFB1) as analyzed by immunoblotting of total protein extracts (30). Heart extracts from tissue-specific TFB1M KO mice (−/−) at 10 weeks of age were used as a control. Tubulin was used as a loading control. (C) Steady-state levels of OXPHOS subunits as determined by immunoblotting of total protein extracts from heart (left) or liver (right) from heterozygous TFB1M KO (+/−), wild-type (+/+) and heterozygous BAC-TFB1M transgenic mice (+/T) at 20 weeks of age. Heart extracts from tissue-specific TFB1M KO mice (−/−) at 10 weeks of age were used as a control. The following subunits were analyzed: Complex I (CI-NDUFB8), Complex II (CII-SDHB), Complex III (CIII-UQCRC2), Complex IV (CIV-MTCOI) and Complex V (CV-ATP5A).
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DDV427F3: Effect of TFB1M overexpression on mitochondria. (A) Quantitative RT–PCR analysis of mtDNA (CoxI) to nuclear DNA (beta-actin) ratios in hearts from wild-type controls (WT) and BAC-TFB1M (C7.1) transgenic mice at 20 weeks of age (n = 3 of each genotype). Error bars represent ±SD. (B) Steady-state levels of TFB1M protein in heart (upper panel) and liver (lower panel) from heterozygous TFB1M KO (+/−), wild-type (+/+) and heterozygous BAC-TFB1M transgenic mice (+/T) and the previously published mouse model overexpressing TFB1M (Tg-mtTFB1) as analyzed by immunoblotting of total protein extracts (30). Heart extracts from tissue-specific TFB1M KO mice (−/−) at 10 weeks of age were used as a control. Tubulin was used as a loading control. (C) Steady-state levels of OXPHOS subunits as determined by immunoblotting of total protein extracts from heart (left) or liver (right) from heterozygous TFB1M KO (+/−), wild-type (+/+) and heterozygous BAC-TFB1M transgenic mice (+/T) at 20 weeks of age. Heart extracts from tissue-specific TFB1M KO mice (−/−) at 10 weeks of age were used as a control. The following subunits were analyzed: Complex I (CI-NDUFB8), Complex II (CII-SDHB), Complex III (CIII-UQCRC2), Complex IV (CIV-MTCOI) and Complex V (CV-ATP5A).
Mentions: Mice that ubiquitously overexpressed TFB1M were created using a bacterial artificial chromosome (BAC) transgenic approach (32–34). We routinely use this approach as it is relatively insensitive to positional effects and results in low-to-moderate ubiquitous overexpression of nucleus-encoded mitochondrially targeted proteins (32–34). The recombinant Tfb1m gene (BAC-TFB1M) and its transcript could be tracked via a silent point mutation that was introduced to abolish an Xma I site in exon 3 (Fig. 2A). The BAC transgenic line C7.1 exhibited ubiquitous TFB1M overexpression levels (Fig. 2B–D) similar to those in the previously published mtTFB1 transgenic mouse line (30) (Fig. 3B) and was selected for further studies. To check for unwanted effects caused by genes flanking Tfb1m in the BAC clone, we generated transgenic mice harboring a version of the BAC clone (BAC-KO) in which Tfb1m expression was silenced by deletion of exon 3. Both the BAC-KO mice and the BAC transgenic C6.1 line, which only showed a minor increase of TFB1M mRNA expression (Fig. 2B), appeared to be completely healthy. A more marked increase in TFB1M expression was observed in the C7.1 line (Fig. 2B–D), and these transgenic mice were subjected to extensive phenotypic analyses. No changes were detected in their metabolism, cardiovascular function, lung function, eye function, grip strength or rotarod performance; similarly, hematological analyses of peripheral blood, broad spectrum clinical chemistry of plasma samples and pathology studies of various tissues showed no evidence of any change that could be related to the C7.1 genotype.Figure 2.

Bottom Line: Non-syndromic deafness and predisposition to aminoglycoside-induced deafness can be caused by specific mutations in the 12S rRNA gene of mtDNA and are thus maternally inherited traits.In contrast, it was recently reported that signaling induced by 'hypermethylation' of two conserved adenosines of 12S rRNA in the mitoribosome is of key pathophysiological importance in sensorineural deafness.We thus conclude that therapies directed against mitoribosomal methylation are unlikely to be beneficial to patients with sensorineural hearing loss or other types of mitochondrial disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Karolinska Institutet, Retzius väg 8, 171 77 Stockholm, Sweden.

No MeSH data available.


Related in: MedlinePlus