Zamora-Dorta, MarcosLaine-Menéndez, SaraAbia, DavidGonzález-García, PilarLópez, Luis CFernández-Montes, PaulaCalvo, EnriqueVázquez, JesúsEnríquez, José AntonioBalsa, Eduardo2025-12-092025-12-092025-06EMBO Rep. 2025 Jun;26(12):3045-3074.https://hdl.handle.net/20.500.12105/26992Defects in mitochondrial oxidative metabolism underlie many genetic disorders with limited treatment options. The incomplete annotation of mitochondrial proteins highlights the need for a comprehensive gene inventory, particularly for Oxidative Phosphorylation (OXPHOS). To address this, we developed a CRISPR/Cas9 loss-of-function library targeting nuclear-encoded mitochondrial genes and conducted galactose-based screenings to identify novel regulators of mitochondrial function. Our study generates a gene catalog essential for mitochondrial metabolism and maps a dynamic network of mitochondrial pathways, focusing on OXPHOS complexes. Computational analysis identifies RTN4IP1 and ECHS1 as key OXPHOS genes linked to mitochondrial diseases in humans. RTN4IP1 is 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. Its deletion impairs branched-chain amino acids (BCAA) catabolism, disrupting 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.We thank all the members of the Balsa laboratory for discussions regarding this project. Dr. Luis C López (University of Granada) kindly provided fibroblasts from a healthy donor and a COQ7-mutated patient. Dra. Cristina Ugalde (Fundación de Investigación Hospital 12 de Octubre, imas12) generously provided 143B and HeLa cells. This work was supported by grants to Dr. Balsa from the Spanish Government, Ministerio de Ciencia e Innovación (PID2019-110766GA-I00 and PID2022-137404OB-I00), European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation (ERC-2020-STG grant agreement n° 948478), and Fundación CRIS contra el cancer (PR_EX_2022-01). The CBM is supported by Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, and is a Severo Ochoa Center of Excellence (grant CEX2021-001154-S) funded by MICIN/AEI/10.13039/501100011033. MZD was supported in part by an FPIUAM PhD fellowship. SLM was supported in part by a Margarita Salas-UAM postdoctoral fellowship (CA4/RSUE/2022-00037). JAE is supported by PID2021-1279880B funded by MICINN/AEI/10.13039/501100011033 and the European Union “NextGenerationEU”/Plan de Recuperación Transformación y Resiliencia -PRTR; “la Caixa” Banking Foundation (project LCF/PR/H23/ 52430010) and CIBERFES (CB16/10/00282) funded by ISCIII. PFM is supported by FPU21/06416 by a fellowship. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MICINN), and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033).engVoRhttp://creativecommons.org/licenses/by/4.0/CRISPR ScreeningECHS1MitochondriaOXPHOSRTN4IP1Attribution 4.0 International4030157226(12)3045-3074open access