Using mitoribosomal profiling to investigate human mitochondrial translation [version 2; referees: 2 approved]
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-tRNAPhe whilst in...
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| Format: | Article |
| Language: | English |
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Wellcome
2018-01-01
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| Series: | Wellcome Open Research |
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| Online Access: | https://wellcomeopenresearch.org/articles/2-116/v2 |
| _version_ | 1818977429434138624 |
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| author | Fei Gao Maria Wesolowska Reuven Agami Koos Rooijers Fabricio Loayza-Puch Conor Lawless Robert N. Lightowlers Zofia M. A. Chrzanowska-Lightowlers |
| author_facet | Fei Gao Maria Wesolowska Reuven Agami Koos Rooijers Fabricio Loayza-Puch Conor Lawless Robert N. Lightowlers Zofia M. A. Chrzanowska-Lightowlers |
| author_sort | Fei Gao |
| collection | DOAJ |
| 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-tRNAPhe whilst in humans it is mt-tRNAVal. We have previously shown that when a mutation in mt-tRNAVal causes very low steady state levels, there is preferential recruitment of mt-tRNAPhe. 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-tRNAPhe. 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-tRNAPhe containing mitoribosomes with those of controls that integrate mt-tRNAVal 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-tRNAVal cell line suggest that despite mt-tRNAPhe being preferred in the porcine mitoribosome, its integration into the human counterpart results in a suboptimal structure that modifies its interaction with mt-mRNAs. |
| first_indexed | 2024-12-20T16:27:36Z |
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| id | doaj.art-82e3c64244ab4623b506bdd6b8560c7b |
| institution | Directory Open Access Journal |
| issn | 2398-502X |
| language | English |
| last_indexed | 2024-12-20T16:27:36Z |
| publishDate | 2018-01-01 |
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| spelling | doaj.art-82e3c64244ab4623b506bdd6b8560c7b2022-12-21T19:33:22ZengWellcomeWellcome Open Research2398-502X2018-01-01210.12688/wellcomeopenres.13119.214957Using mitoribosomal profiling to investigate human mitochondrial translation [version 2; referees: 2 approved]Fei Gao0Maria Wesolowska1Reuven Agami2Koos Rooijers3Fabricio Loayza-Puch4Conor Lawless5Robert N. Lightowlers6Zofia M. A. Chrzanowska-Lightowlers7The Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UKImmunocore Ltd, Oxford, UKThe Netherlands Cancer Institute, Amsterdam, NetherlandsHubrecht Institute, Utrecht, NetherlandsThe Netherlands Cancer Institute, Amsterdam, NetherlandsThe Wellcome Centre for Mitochondrial Research, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UKThe Wellcome Centre for Mitochondrial Research, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UKThe Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UKBackground: 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-tRNAPhe whilst in humans it is mt-tRNAVal. We have previously shown that when a mutation in mt-tRNAVal causes very low steady state levels, there is preferential recruitment of mt-tRNAPhe. 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-tRNAPhe. 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-tRNAPhe containing mitoribosomes with those of controls that integrate mt-tRNAVal 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-tRNAVal cell line suggest that despite mt-tRNAPhe being preferred in the porcine mitoribosome, its integration into the human counterpart results in a suboptimal structure that modifies its interaction with mt-mRNAs.https://wellcomeopenresearch.org/articles/2-116/v2Experimental Biophysical MethodsGenomics |
| spellingShingle | Fei Gao Maria Wesolowska Reuven Agami Koos Rooijers Fabricio Loayza-Puch Conor Lawless Robert N. Lightowlers Zofia M. A. Chrzanowska-Lightowlers Using mitoribosomal profiling to investigate human mitochondrial translation [version 2; referees: 2 approved] Wellcome Open Research Experimental Biophysical Methods Genomics |
| title | Using mitoribosomal profiling to investigate human mitochondrial translation [version 2; referees: 2 approved] |
| title_full | Using mitoribosomal profiling to investigate human mitochondrial translation [version 2; referees: 2 approved] |
| title_fullStr | Using mitoribosomal profiling to investigate human mitochondrial translation [version 2; referees: 2 approved] |
| title_full_unstemmed | Using mitoribosomal profiling to investigate human mitochondrial translation [version 2; referees: 2 approved] |
| title_short | Using mitoribosomal profiling to investigate human mitochondrial translation [version 2; referees: 2 approved] |
| title_sort | using mitoribosomal profiling to investigate human mitochondrial translation version 2 referees 2 approved |
| topic | Experimental Biophysical Methods Genomics |
| url | https://wellcomeopenresearch.org/articles/2-116/v2 |
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