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. 2021 Apr 9;12:2130. doi: 10.1038/s41467-021-22397-5

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a The MOCCI ORF is conserved across vertebrates and contains a predicted transmembrane (TM) domain and the B12D domain found only in NADH-ubiquinone reductase complex 1 MLRQ subunits. The MOCCI transcript is also the host gene for hsa-miR-147b (right), located in the 3′UTR of the transcript. The letters in blue are the sequence of the mature miRNA. b MOCCI is a paralog of NDUFA4 which also contains both the B12D and TM domains. The 3′UTR of the NDUFA4 transcript contains top target site of miR-147b-3p as predicted by TargetScan¹¹⁴, miRDB¹¹⁵, and miRBase¹¹⁶. It is not clear if the binding site in chicken Ndufa4 is functional, as it is not as well conserved. c MOCCI was found in mitochondria-enriched fractions of HEK293T transfected with MOCCI ORF-expressing plasmid (MOCCI-HEK293T). Values given indicates percentage of the total fraction loaded onto the gel. n = 2 biological replicates. Source data are provided as a Source Data file. d Differential solubilization of MOCCI-HEK293T mitochondria indicated that MOCCI was located in the inner membrane (IMM). TOMM70 and MTCO-1 serve as control for outer (OMM) and inner (IMM) mitochondrial membrane proteins, respectively. n = 1 biological replicate. Source data are provided as a Source Data file. e Proteinase K protection assay of MOCCI-HEK293T mitochondria further confirmed IMM localization of MOCCI. n = 1 biological replicate. Source data are provided as a Source Data file. f Blue native PAGE (BN-PAGE) of mouse heart mitochondria with AAV9-mediated MOCCI-FLAG expression (AAV-MOCCI). Probing with α-MOCCI and α-MTCO-1 antibodies, the core enzymatic subunit of CIV, reveal co-migration of MOCCI signal with CIV. AAV-GFP was used as a negative control. Coomassie blue stain of the gel is shown on the right. n = 4 biological replicates. g Two-dimensional BN-SDS-PAGE to confirm co-migration of MOCCI and CIV. The lanes correspond to the bands annotated on the Coomassie blue-stained gel in (f). n = 2 biological replicates. Source data are provided as a Source Data file.