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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

Dimethylation of 3′-terminal loop adenosines in mitochondrial 12S rRNA from BAC-TFB1M transgenic mice. (A) The methylation status of the mitochondrial 12S rRNA was determined by primer extension analysis of total RNA extracts from heart and liver in control (+/+) and BAC-TFB1M transgenic C7.1 (+/T) mice at 20 weeks of age. (B) Relative dimethylation at the A1006 and A1007 residues of 12S rRNA in control (blue bars) and BAC-TFB1M transgenic C7.1 (red bars) mice (n = 3 of each genotype). Error bars represent mean ± SD. (C) Primer extension analysis of 12S rRNA adenosine dimethylation in total RNA extracts from liver. The percentage stop at A1007 (the first nucleotide encountered by reverse transcriptase) corresponds to the degree of dimethylation at this adenosine; the percentage stop at A1006 reveals dimethylation here only in molecules where there is read-through from A1007 (and therefore no dimethylation at A1007); stopping at G1005 occurs upon ddCTP incorporation and represents the percentage of rRNA molecules without dimethylation at A1006 or A1007. The percentages below indicate primer extension stops owing to adenosine dimethylation at A1007 and A1006, as well as lack of adenosine dimethylation, which is evident as transcripts reading through to G1005. Dimethylation at A1006 and/or A1007 was consistently ≥97% in all tissue samples. (D) Primer extension analysis of 12S rRNA adenosine dimethylation in total RNA extracts from heart. Total RNA extracts from heart of homozygous TFB1M tissue-specific KO mice (−/−) at 10 weeks of age were used as a control. The percentages below indicate primer extension stops owing to adenosine dimethylation at A1007 and A1006, as well as lack of adenosine dimethylation, which is evident as transcripts reading through to G1005. With the exception of the homozygous TFB1M knockout in heart (−/−), dimethylation at A1006 and/or A1007 was consistently ≥97% in all tissue samples. Thus the previous report of lower methylation levels (30) was not corroborated in our reanalysis of the same tissue samples (Tg-mtTFB1).
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DDV427F4: Dimethylation of 3′-terminal loop adenosines in mitochondrial 12S rRNA from BAC-TFB1M transgenic mice. (A) The methylation status of the mitochondrial 12S rRNA was determined by primer extension analysis of total RNA extracts from heart and liver in control (+/+) and BAC-TFB1M transgenic C7.1 (+/T) mice at 20 weeks of age. (B) Relative dimethylation at the A1006 and A1007 residues of 12S rRNA in control (blue bars) and BAC-TFB1M transgenic C7.1 (red bars) mice (n = 3 of each genotype). Error bars represent mean ± SD. (C) Primer extension analysis of 12S rRNA adenosine dimethylation in total RNA extracts from liver. The percentage stop at A1007 (the first nucleotide encountered by reverse transcriptase) corresponds to the degree of dimethylation at this adenosine; the percentage stop at A1006 reveals dimethylation here only in molecules where there is read-through from A1007 (and therefore no dimethylation at A1007); stopping at G1005 occurs upon ddCTP incorporation and represents the percentage of rRNA molecules without dimethylation at A1006 or A1007. The percentages below indicate primer extension stops owing to adenosine dimethylation at A1007 and A1006, as well as lack of adenosine dimethylation, which is evident as transcripts reading through to G1005. Dimethylation at A1006 and/or A1007 was consistently ≥97% in all tissue samples. (D) Primer extension analysis of 12S rRNA adenosine dimethylation in total RNA extracts from heart. Total RNA extracts from heart of homozygous TFB1M tissue-specific KO mice (−/−) at 10 weeks of age were used as a control. The percentages below indicate primer extension stops owing to adenosine dimethylation at A1007 and A1006, as well as lack of adenosine dimethylation, which is evident as transcripts reading through to G1005. With the exception of the homozygous TFB1M knockout in heart (−/−), dimethylation at A1006 and/or A1007 was consistently ≥97% in all tissue samples. Thus the previous report of lower methylation levels (30) was not corroborated in our reanalysis of the same tissue samples (Tg-mtTFB1).

Mentions: High levels of dimethylated A1006 and/or A1007 (>97%) were measured in 12S rRNA from liver and heart of BAC-TFB1M (C7.1) and littermate control mice (Fig. 4A and B). Similarly, samples from the previously published Tg-mtTFB1 mouse strain (30) showed, in our hands, near complete (∼97%) 12S rRNA dimethylation that did not differ from littermate control mice in liver (Fig. 4C) and heart (Fig. 4D). We proceeded to isolate total RNA from liver and heart also from tissue-specific homozygous TFB1M knockout (KO) mice (−/−), heterozygous TFB1M KO mice (+/−), wild-type mice (+/+), BAC-TFB1M mice and BAC-KO mice. With the exception of the homozygous TFB1M heart knockout, there was no difference in the methylation status of 12S rRNA (Fig. 4C and D). The heart-specific TFB1M knockouts developed a progressive cardiomyopathy and, consistent with a previous study (17), by the age of 10 weeks <40% of the 12S rRNA in the heart tissue remained dimethylated (Fig. 4D).Figure 4.


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)

Dimethylation of 3′-terminal loop adenosines in mitochondrial 12S rRNA from BAC-TFB1M transgenic mice. (A) The methylation status of the mitochondrial 12S rRNA was determined by primer extension analysis of total RNA extracts from heart and liver in control (+/+) and BAC-TFB1M transgenic C7.1 (+/T) mice at 20 weeks of age. (B) Relative dimethylation at the A1006 and A1007 residues of 12S rRNA in control (blue bars) and BAC-TFB1M transgenic C7.1 (red bars) mice (n = 3 of each genotype). Error bars represent mean ± SD. (C) Primer extension analysis of 12S rRNA adenosine dimethylation in total RNA extracts from liver. The percentage stop at A1007 (the first nucleotide encountered by reverse transcriptase) corresponds to the degree of dimethylation at this adenosine; the percentage stop at A1006 reveals dimethylation here only in molecules where there is read-through from A1007 (and therefore no dimethylation at A1007); stopping at G1005 occurs upon ddCTP incorporation and represents the percentage of rRNA molecules without dimethylation at A1006 or A1007. The percentages below indicate primer extension stops owing to adenosine dimethylation at A1007 and A1006, as well as lack of adenosine dimethylation, which is evident as transcripts reading through to G1005. Dimethylation at A1006 and/or A1007 was consistently ≥97% in all tissue samples. (D) Primer extension analysis of 12S rRNA adenosine dimethylation in total RNA extracts from heart. Total RNA extracts from heart of homozygous TFB1M tissue-specific KO mice (−/−) at 10 weeks of age were used as a control. The percentages below indicate primer extension stops owing to adenosine dimethylation at A1007 and A1006, as well as lack of adenosine dimethylation, which is evident as transcripts reading through to G1005. With the exception of the homozygous TFB1M knockout in heart (−/−), dimethylation at A1006 and/or A1007 was consistently ≥97% in all tissue samples. Thus the previous report of lower methylation levels (30) was not corroborated in our reanalysis of the same tissue samples (Tg-mtTFB1).
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DDV427F4: Dimethylation of 3′-terminal loop adenosines in mitochondrial 12S rRNA from BAC-TFB1M transgenic mice. (A) The methylation status of the mitochondrial 12S rRNA was determined by primer extension analysis of total RNA extracts from heart and liver in control (+/+) and BAC-TFB1M transgenic C7.1 (+/T) mice at 20 weeks of age. (B) Relative dimethylation at the A1006 and A1007 residues of 12S rRNA in control (blue bars) and BAC-TFB1M transgenic C7.1 (red bars) mice (n = 3 of each genotype). Error bars represent mean ± SD. (C) Primer extension analysis of 12S rRNA adenosine dimethylation in total RNA extracts from liver. The percentage stop at A1007 (the first nucleotide encountered by reverse transcriptase) corresponds to the degree of dimethylation at this adenosine; the percentage stop at A1006 reveals dimethylation here only in molecules where there is read-through from A1007 (and therefore no dimethylation at A1007); stopping at G1005 occurs upon ddCTP incorporation and represents the percentage of rRNA molecules without dimethylation at A1006 or A1007. The percentages below indicate primer extension stops owing to adenosine dimethylation at A1007 and A1006, as well as lack of adenosine dimethylation, which is evident as transcripts reading through to G1005. Dimethylation at A1006 and/or A1007 was consistently ≥97% in all tissue samples. (D) Primer extension analysis of 12S rRNA adenosine dimethylation in total RNA extracts from heart. Total RNA extracts from heart of homozygous TFB1M tissue-specific KO mice (−/−) at 10 weeks of age were used as a control. The percentages below indicate primer extension stops owing to adenosine dimethylation at A1007 and A1006, as well as lack of adenosine dimethylation, which is evident as transcripts reading through to G1005. With the exception of the homozygous TFB1M knockout in heart (−/−), dimethylation at A1006 and/or A1007 was consistently ≥97% in all tissue samples. Thus the previous report of lower methylation levels (30) was not corroborated in our reanalysis of the same tissue samples (Tg-mtTFB1).
Mentions: High levels of dimethylated A1006 and/or A1007 (>97%) were measured in 12S rRNA from liver and heart of BAC-TFB1M (C7.1) and littermate control mice (Fig. 4A and B). Similarly, samples from the previously published Tg-mtTFB1 mouse strain (30) showed, in our hands, near complete (∼97%) 12S rRNA dimethylation that did not differ from littermate control mice in liver (Fig. 4C) and heart (Fig. 4D). We proceeded to isolate total RNA from liver and heart also from tissue-specific homozygous TFB1M knockout (KO) mice (−/−), heterozygous TFB1M KO mice (+/−), wild-type mice (+/+), BAC-TFB1M mice and BAC-KO mice. With the exception of the homozygous TFB1M heart knockout, there was no difference in the methylation status of 12S rRNA (Fig. 4C and D). The heart-specific TFB1M knockouts developed a progressive cardiomyopathy and, consistent with a previous study (17), by the age of 10 weeks <40% of the 12S rRNA in the heart tissue remained dimethylated (Fig. 4D).Figure 4.

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