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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...

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Autores principales: Potter, Alisa, Cabrera-Orefice, Alfredo, Spelbrink, Johannes N
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2023
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.
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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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