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Using mitoribosomal profiling to investigate human mitochondrial translation
Background: Gene expression in human mitochondria has various idiosyncratic features. One of these was recently revealed as the unprecedented recruitment of a mitochondrially-encoded tRNA as a structural component of the large mitoribosomal subunit. In porcine particles this is mt-tRNA (Phe) whilst...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
F1000 Research Limited
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771143/ https://www.ncbi.nlm.nih.gov/pubmed/29387808 http://dx.doi.org/10.12688/wellcomeopenres.13119.2 |
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author | Gao, Fei Wesolowska, Maria Agami, Reuven Rooijers, Koos Loayza-Puch, Fabricio Lawless, Conor Lightowlers, Robert N. Chrzanowska-Lightowlers, Zofia M. A. |
author_facet | Gao, Fei Wesolowska, Maria Agami, Reuven Rooijers, Koos Loayza-Puch, Fabricio Lawless, Conor Lightowlers, Robert N. Chrzanowska-Lightowlers, Zofia M. A. |
author_sort | Gao, Fei |
collection | PubMed |
description | Background: Gene expression in human mitochondria has various idiosyncratic features. One of these was recently revealed as the unprecedented recruitment of a mitochondrially-encoded tRNA as a structural component of the large mitoribosomal subunit. In porcine particles this is mt-tRNA (Phe) whilst in humans it is mt-tRNA (Val). We have previously shown that when a mutation in mt-tRNA (Val) causes very low steady state levels, there is preferential recruitment of mt-tRNA (Phe). We have investigated whether this altered mitoribosome affects intra-organellar protein synthesis. Methods: By using mitoribosomal profiling we have revealed aspects of mitoribosome behaviour with its template mt-mRNA under both normal conditions as well as those where the mitoribosome has incorporated mt-tRNA (Phe). Results: Analysis of the mitoribosome residency on transcripts under control conditions reveals that although mitochondria employ only 22 mt-tRNAs for protein synthesis, the use of non-canonical wobble base pairs at codon position 3 does not cause any measurable difference in mitoribosome occupancy irrespective of the codon. Comparison of the profile of aberrant mt-tRNA (Phe) containing mitoribosomes with those of controls that integrate mt-tRNA (Val) revealed that the impaired translation seen in the latter was not due to stalling on triplets encoding either of these amino acids. The alterations in mitoribosome interactions with start codons was not directly attributable to the either the use of non-cognate initiation codons or the presence or absence of 5’ leader sequences, except in the two bicistronic RNA units, RNA7 and RNA14 where the initiation sites are internal. Conclusions: These data report the power of mitoribosomal profiling in helping to understand the subtleties of mammalian mitochondrial protein synthesis. Analysis of profiles from the mutant mt-tRNA (Val) cell line suggest that despite mt-tRNA (Phe) being preferred in the porcine mitoribosome, its integration into the human counterpart results in a suboptimal structure that modifies its interaction with mt-mRNAs. |
format | Online Article Text |
id | pubmed-5771143 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | F1000 Research Limited |
record_format | MEDLINE/PubMed |
spelling | pubmed-57711432018-01-29 Using mitoribosomal profiling to investigate human mitochondrial translation Gao, Fei Wesolowska, Maria Agami, Reuven Rooijers, Koos Loayza-Puch, Fabricio Lawless, Conor Lightowlers, Robert N. Chrzanowska-Lightowlers, Zofia M. A. Wellcome Open Res Research Article Background: Gene expression in human mitochondria has various idiosyncratic features. One of these was recently revealed as the unprecedented recruitment of a mitochondrially-encoded tRNA as a structural component of the large mitoribosomal subunit. In porcine particles this is mt-tRNA (Phe) whilst in humans it is mt-tRNA (Val). We have previously shown that when a mutation in mt-tRNA (Val) causes very low steady state levels, there is preferential recruitment of mt-tRNA (Phe). We have investigated whether this altered mitoribosome affects intra-organellar protein synthesis. Methods: By using mitoribosomal profiling we have revealed aspects of mitoribosome behaviour with its template mt-mRNA under both normal conditions as well as those where the mitoribosome has incorporated mt-tRNA (Phe). Results: Analysis of the mitoribosome residency on transcripts under control conditions reveals that although mitochondria employ only 22 mt-tRNAs for protein synthesis, the use of non-canonical wobble base pairs at codon position 3 does not cause any measurable difference in mitoribosome occupancy irrespective of the codon. Comparison of the profile of aberrant mt-tRNA (Phe) containing mitoribosomes with those of controls that integrate mt-tRNA (Val) revealed that the impaired translation seen in the latter was not due to stalling on triplets encoding either of these amino acids. The alterations in mitoribosome interactions with start codons was not directly attributable to the either the use of non-cognate initiation codons or the presence or absence of 5’ leader sequences, except in the two bicistronic RNA units, RNA7 and RNA14 where the initiation sites are internal. Conclusions: These data report the power of mitoribosomal profiling in helping to understand the subtleties of mammalian mitochondrial protein synthesis. Analysis of profiles from the mutant mt-tRNA (Val) cell line suggest that despite mt-tRNA (Phe) being preferred in the porcine mitoribosome, its integration into the human counterpart results in a suboptimal structure that modifies its interaction with mt-mRNAs. F1000 Research Limited 2018-01-29 /pmc/articles/PMC5771143/ /pubmed/29387808 http://dx.doi.org/10.12688/wellcomeopenres.13119.2 Text en Copyright: © 2018 Gao F et al. http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Gao, Fei Wesolowska, Maria Agami, Reuven Rooijers, Koos Loayza-Puch, Fabricio Lawless, Conor Lightowlers, Robert N. Chrzanowska-Lightowlers, Zofia M. A. Using mitoribosomal profiling to investigate human mitochondrial translation |
title | Using mitoribosomal profiling to investigate human mitochondrial translation |
title_full | Using mitoribosomal profiling to investigate human mitochondrial translation |
title_fullStr | Using mitoribosomal profiling to investigate human mitochondrial translation |
title_full_unstemmed | Using mitoribosomal profiling to investigate human mitochondrial translation |
title_short | Using mitoribosomal profiling to investigate human mitochondrial translation |
title_sort | using mitoribosomal profiling to investigate human mitochondrial translation |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771143/ https://www.ncbi.nlm.nih.gov/pubmed/29387808 http://dx.doi.org/10.12688/wellcomeopenres.13119.2 |
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