Time-resolved mitochondrial-focused screening identifies regulatory components of oxidative metabolism
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (Arcadia Science)
Abstract
Defects in mitochondrial oxidative metabolism contribute to various genetic inherited disorders, termed mitochondrial diseases, with limited treatment options. Given the lack of functional annotation for numerous mitochondrial proteins, there is a necessity for an extensive gene inventory related to mitochondrial function, with special interest in Oxidative Phosphorylation (OXPHOS). To address this gap, we developed a CRISPR/Cas9 loss-of-function library targeting nuclear-encoded mitochondrial genes and conducted galactose-based screenings at various time points to uncover novel regulators of mitochondrial function. Our study resulted in a gene catalog essential for mitochondrial oxidative metabolism, and constructed a dynamic timeline mapping a broad network of mitochondrial pathways, with a particular focus on the OXPHOS complexes. Computational analysis pinpointed RTN4IP1 and ECHS1 as key genes strongly associated with OXPHOS and whose mutations are associated with mitochondrial diseases in humans. RTN4IP1 was found to be crucial for mitochondrial respiration, with complexome profiling revealing its role as an assembly factor required for the complete assembly of complex I. Furthermore, we discovered that ECHS1 controls oxidative metabolism independently of its canonical function in fatty acid oxidation. Deletion of ECHS1 leads to reduced catabolism of branched-chain amino acids (BCAAs), which impairs the activity of lipoic acid-dependent enzymes such as pyruvate dehydrogenase (PDH). This deleterious phenotype can be rescued by restricting valine intake or catabolism in ECHS1-deficient cells.
Article activity feed
-
Whether RTN4IP1 functions through interactions with other partners or by facilitating posttranslational modifications during CI assembly remains unanswered and warrants further investigation
Do you have thoughts on how you would further experimentally decouple the Complex I assembly and CoQ biosynthesis functions of RTN4IP1?
-
Pathway analysis revealed that the affected biological processes of upregulated proteins were related to cell-substrate adhesion and extracellular organization
Did you look into why RTN4IP1 upregulated cell adhesion and extracellular organization factors? This is surprising to me given the clear role you have demonstrated in Complex I assembly.
-
This phenomenon was particularly evident in the context of CI, where the identification of assembly factors increased nearly threefold between 24 hours and 1 week
Do you have a hypothesis as to why the galactose growth phenotype is particularly pronounced for Complex I assembly factors? Is complex I especially reliant on assembly factors to adopt its active configuration?
-
Dynamics and surveillance, along with the Signaling pathway, exhibited notable enrichment in genes with no discernible phenotype, implying that genes within these categories may be dispensable for proliferation in galactose, likely owing to their limited influence on OXPHOS activity
Is it possible to use an alternative functional genomics approach to study genes involved in dynamics and signaling? How would you set up such a screen?
-
Even though we now possess a very detailed picture of the core proteins that comprise the OXPHOS complexes, the list of assembly factors and regulatory components is far from being completed
You mention that many nuclear-encoded genes important in mitochondrial function have not yet been identified. Is it possible to adapt your screening approach to identify these genes without having to use a genome-wide library?
-
