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Let's make it clear: systematic exploration of mitochondrial DNA– and RNA–protein complexes by complexome profiling
Complexome profiling (CP) is a powerful tool for systematic investigation of protein interactors that has been primarily applied to study the composition and dynamics of mitochondrial protein complexes. Here, we further optimized this method to extend its application to survey mitochondrial DNA- and...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10602928/ https://www.ncbi.nlm.nih.gov/pubmed/37615582 http://dx.doi.org/10.1093/nar/gkad697 |
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author | Potter, Alisa Cabrera-Orefice, Alfredo Spelbrink, Johannes N |
author_facet | Potter, Alisa Cabrera-Orefice, Alfredo Spelbrink, Johannes N |
author_sort | Potter, Alisa |
collection | PubMed |
description | Complexome profiling (CP) is a powerful tool for systematic investigation of protein interactors that has been primarily applied to study the composition and dynamics of mitochondrial protein complexes. Here, we further optimized this method to extend its application to survey mitochondrial DNA- and RNA-interacting protein complexes. We established that high-resolution clear native gel electrophoresis (hrCNE) is a better alternative to preserve DNA– and RNA–protein interactions that are otherwise disrupted when samples are separated by the widely used blue native gel electrophoresis (BNE). In combination with enzymatic digestion of DNA, our CP approach improved the identification of a wide range of protein interactors of the mitochondrial gene expression system without compromising the detection of other multiprotein complexes. The utility of this approach was particularly demonstrated by analysing the complexome changes in human mitochondria with impaired gene expression after transient, chemically induced mitochondrial DNA depletion. Effects of RNase on mitochondrial protein complexes were also evaluated and discussed. Overall, our adaptations significantly improved the identification of mitochondrial DNA– and RNA–protein interactions by CP, thereby unlocking the comprehensive analysis of a near-complete mitochondrial complexome in a single experiment. |
format | Online Article Text |
id | pubmed-10602928 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-106029282023-10-28 Let's make it clear: systematic exploration of mitochondrial DNA– and RNA–protein complexes by complexome profiling Potter, Alisa Cabrera-Orefice, Alfredo Spelbrink, Johannes N Nucleic Acids Res RNA and RNA-protein complexes Complexome profiling (CP) is a powerful tool for systematic investigation of protein interactors that has been primarily applied to study the composition and dynamics of mitochondrial protein complexes. Here, we further optimized this method to extend its application to survey mitochondrial DNA- and RNA-interacting protein complexes. We established that high-resolution clear native gel electrophoresis (hrCNE) is a better alternative to preserve DNA– and RNA–protein interactions that are otherwise disrupted when samples are separated by the widely used blue native gel electrophoresis (BNE). In combination with enzymatic digestion of DNA, our CP approach improved the identification of a wide range of protein interactors of the mitochondrial gene expression system without compromising the detection of other multiprotein complexes. The utility of this approach was particularly demonstrated by analysing the complexome changes in human mitochondria with impaired gene expression after transient, chemically induced mitochondrial DNA depletion. Effects of RNase on mitochondrial protein complexes were also evaluated and discussed. Overall, our adaptations significantly improved the identification of mitochondrial DNA– and RNA–protein interactions by CP, thereby unlocking the comprehensive analysis of a near-complete mitochondrial complexome in a single experiment. Oxford University Press 2023-08-24 /pmc/articles/PMC10602928/ /pubmed/37615582 http://dx.doi.org/10.1093/nar/gkad697 Text en © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | RNA and RNA-protein complexes Potter, Alisa Cabrera-Orefice, Alfredo Spelbrink, Johannes N Let's make it clear: systematic exploration of mitochondrial DNA– and RNA–protein complexes by complexome profiling |
title | Let's make it clear: systematic exploration of mitochondrial DNA– and RNA–protein complexes by complexome profiling |
title_full | Let's make it clear: systematic exploration of mitochondrial DNA– and RNA–protein complexes by complexome profiling |
title_fullStr | Let's make it clear: systematic exploration of mitochondrial DNA– and RNA–protein complexes by complexome profiling |
title_full_unstemmed | Let's make it clear: systematic exploration of mitochondrial DNA– and RNA–protein complexes by complexome profiling |
title_short | Let's make it clear: systematic exploration of mitochondrial DNA– and RNA–protein complexes by complexome profiling |
title_sort | let's make it clear: systematic exploration of mitochondrial dna– and rna–protein complexes by complexome profiling |
topic | RNA and RNA-protein complexes |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10602928/ https://www.ncbi.nlm.nih.gov/pubmed/37615582 http://dx.doi.org/10.1093/nar/gkad697 |
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