Unprocessed RNA intermediates interfere with mitochondrial translation and cause respiratory chain deficiency. R. Kopajtich1, T. B. Haack1,2, P. Freisinger3, T. Wieland1,2, J. Rorbach4, T. J. Nicholls4, E. Baruffini5, A. Walther1,2, K. Danhauser1, F. A. Zimmermann6, R. A. Husain7, H. Mundy8, I. Ferrero5, T. M. Strom1,2, T. Meitinger1,2, R. W. Taylor9, M. Minczuk4, J. A. Mayr6, H. Prokisch1,2 1) Institute of Human Genetics, Helmholtz Zentrum München, Munich / Neuherberg, Germany; 2) Institute of Human Genetics, Techniche Universität München, Munich, Germany; 3) Department of Pediatrics, Klinikum Reutlingen, Reutlingen, Germany; 4) MRC Mitochondrial Biology Unit, Cambridge, United Kingdom; 5) Department of Life Sciences, University of Parma, Parma, Italy; 6) Department of Pediatrics, Paracelsus Medical University Salzburg, Salzburg, Austria; 7) Department of Neuropediatrics, Jena University Hospital, Jena, Germany; 8) Centre for Inherited Metabolic Disease, Evelina Children's Hospital, Guys and St Thomas' NHS Foundation Trust, London, United Kingdom; 9) Wellcome Trust Centre for Mitochondrial Research, Institute for Ageing and Health, The Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom.

   Mitochondria harbor their own genome, transcription and translation machinery. The human mitochondrial genome encodes 13 mRNAs, 22 tRNAs and 2 rRNAs to produce 13 subunits of the respiratory chain. Transcription of the mitochondrial DNA produces large polycistronic precursor transcripts that have to be precisely processed since most genes are directly adjacent or separated by only a few non-coding nucleotides. Most human mRNA and rRNA genes are flanked by tRNA genes and, according to the 't-RNA punctuation model', excision of these tRNAs leads to the liberation of the corresponding mRNAs and rRNAs. The primary transcript is processed by subsequent cleavage of RNase P and RNase Z, encoded by MRPP1,2,3 and ELAC2 respectively. By exome sequencing and subsequent mutation screening, we now identified 5 individuals from 3 different families carrying either compound heterozygous or homozygous mutations in ELAC2. All patients presented with infancy-onset hypertrophic cardiomyopathy, lactic acidosis and respiratory chain defects. Quantitative PCR demonstrated the accumulation of unprocessed RNA intermediates up to 400-fold in in skeletal muscle samples and up to 30-fold in fibroblasts. However, Northern blot analysis showed no change in steady state levels of rRNAs, mRNAs or tRNAs, and tRNAs were found to be existing in their mature form including the trinucleotide CCA 3end. Nevertheless, the patients cell lines displayed an impaired mitochondrial translation. In contrast to prior studies the translation defect could not be explained by changes in the abundance of individual RNA species. Finally, transcriptome analysis by RNAseq in fibroblasts showed that approximately 10% of the mitochondrial mRNAs still have a tRNA attached to their 5-ends. Therefore we propose a model in which aberrantly processed mRNA species (with an extended 5-terminus) fail to be effectively eliminated by the RNA surveillance machinery interfere with translation. This constitutes a new pathomechanism for mitochondrial disorders. This study highlights that next generation sequencing not only provided a molecular diagnosis for these patients but also helped to understand the underlying pathomechanism.

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