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 optimised 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 multi-protein 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 mtDNA 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.
Author: | Alisa PotterORCiD, Alfredo Cabrera-OreficeORCiD, Johannes N. SpelbrinkORCiD |
---|---|
URN: | urn:nbn:de:hebis:30:3-735457 |
DOI: | https://doi.org/10.1101/2023.04.03.534993 |
Parent Title (English): | bioRxiv |
Document Type: | Article |
Language: | English |
Date of Publication (online): | 2023/04/03 |
Date of first Publication: | 2023/04/03 |
Publishing Institution: | Universitätsbibliothek Johann Christian Senckenberg |
Release Date: | 2023/05/25 |
Issue: | 2023.04.03.534993 |
Page Number: | 37 |
Institutes: | Medizin / Medizin |
Dewey Decimal Classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit | |
Sammlungen: | Universitätspublikationen |
Licence (German): | Creative Commons - CC BY-NC-ND - Namensnennung - Nicht kommerziell - Keine Bearbeitungen 4.0 International |