O'Sullivan 2015 Abstract MiPschool London 2015

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Role of TFB1M-mediated 12S m62A ribosomal RNA methylation in m.1555A>G-related hearing loss.

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O’Sullivan M, Rutland P, Lucas D, Ashton E, Hendricks S, Rahman S, Bitner-Glindzicz M (2015)

Event: MiPschool London 2015

One in 500 people has a maternally inherited mitochondrial DNA (mtDNA) mutation, termed m.1555A>G, which predisposes to extreme hypersensitivity to aminoglycoside antibiotics, resulting in hearing loss [1]. Twenty years on from the discovery of the m.1555A>G mutation, the fundamental mechanism of ribosomal dysfunction is unclear and two discordant mechanisms have been postulated - ribosomal mRNA misreading and 12S 12S m62A ribosomal RNA (rRNA) methylation [2]. The long-standing concept of mRNA misreading suggests that the fidelity of tRNA selection during mitochondrial translation is impaired leading to misincorporation of amino acids. Recent evidence from cell and animal models indicates that the m.1555A>G mutation may make the 12S rRNA transcript a better substrate for 12S m62A methylation by the mitochondrial transcription factor B1 (TFB1M) enzyme during ribosome biogenesis and trigger so-called ‘hypermethylation’.

The emerging concept of hypermethylation has yet to be investigated in RNA from patients with m.1555A>G. 12S 12S m62A rRNA methylation, occurring at two successive nucleotides (m.1584A and m.1583A) in close proximity to m.1555A>G, was examined using a novel fluorescent primer extension assay, designed for use in blood samples from paediatric patients. The primer extension method of detecting an RNA modification is based on the principles of reverse transcriptase (RT) mediated cDNA synthesis. First, a 5’ end labelled DNA oligonucleotide primer hybridises to a sequence downstream (3’ end) of m62A. Second, RT extends the hybridised primer. The presence of 12S m62A halts RT. In the absence of 12S m62A, primer extension continues and is halted downstream of the 12S m62A modification site by incorporation of a ddNTP nucleotide in the reaction mix (as per Sanger sequencing). Capillary electrophoresis separates fragment populations.

We examined m62A methylation in 14 patients with m.1555A>G, and controls, and found all detectable 12S rRNA transcripts to be fully methylated in both groups [2]. Moreover, different RNA samples derived from the same patient (lymphocyte, fibroblast and lymphoblastoid) revealed that only transformed cells contained some unmethylated 12S rRNA transcripts, with all detectable 12S rRNA transcripts derived from primary samples being completely 12S m62A-methylated [2].


Labels: MiParea: mtDNA;mt-genetics, Patients  Pathology: Aging;senescence 


Tissue;cell: Blood cells, Fibroblast, Lymphocyte 





Abstract contiued

Our data indicate that previous findings may be an artefact of the experimental models used to study this hypothesis and hypermethylation is unlikely to be a pathogenic mechanism of m.1555A>G-related hearing loss [2]. This has three important implications. First, mRNA misreading remains as the most likely mechanistic concept to explain ribosomal dysfunction related to m.1555A>G. Second, it appears that RNA methylation patterns observed in cell and animal models may not be always representative of the human situation. Finally, the novel fluorescent-based primer extension assay used in this work may be relevant to the study of other mitochondrial disorders. For example, the 12S 12S m62A rRNA modification has been linked to type II diabetes, and the transfer RNALeu(UUR) τm5U wobble modification has been associated with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes [3,4].

Affiliations

1-Genetics and Genomic Medicine, UCL Inst Child Health, London, UK. - sejjosu@live.ucl.ac.uk

2-Nuffield Hearing and Speech Centre, Royal National Throat Nose Ear Hospital, London

3-NE Thames Regional Genetics Service, London

4-Metabolic Dept, Great Ormond Street Hospital Children NHS Trust, London

5-Barnet Chase Farm Hospitals NHS Trust, Enfield, Middlesex

6-Centre Auditory Research, UCL Ear Institute, London


References

  1. Bitner-Glindzicz M, Pembrey M, Duncan A, Heron J, Ring SM, Hall A, Rahman S (2009) Prevalence of mitochondrial 1555A>G mutation in European children. N Engl J Med 360:640–2.
  2. O’Sullivan M, Rutland P, Lucas D, Ashton E, Hendricks S, Rahman S, and Bitner-Glindzicz M (2014) Mitochondrial m.1584A 12S m62A rRNA methylation in families with m.1555A>G associated hearing loss. Hum Mol Genet 24:1036-44.
  3. Sharoyko VV, Abels M, Sun J, Nicholas LM, Mollet IG, Stamenkovic JA, Göhring I, Malmgren S, Storm P, Fadista J, Spégel P, Metodiev MD, Larsson NG, Eliasson L, Wierup N, Mulder H (2014) Loss of TFB1M results in mitochondrial dysfunction that leads to impaired insulin secretion and diabetes. Hum Mol Genet 23: 5733-49.
  4. Kirino Y, Goto YI, Campos Y, Arenas J, Suzuki T (2005) Specific correlation between the wobble modification deficiency in mutant tRNAs and the clinical features of a human mitochondrial disease. Proc Natl Acad Sci USA 102: 7127–32.