![]() Enrichment and P-values were calculated using DESeq2. ( E) Enrichment plots for heavy- and light-strand FASTKD2-GFP crosslink sites. Enrichment of light-strand transcripts is indicated in blue, enrichment of heavy-strand transcripts is indicated in red. ( D) Heat map representing significant (false discovery rate <0.05) RNA crosslink sites in MLS-GFP and FASTKD2-GFP libraries. Crosslink site density was calculated using the sum of annotated gene lengths for each chromosome. ( C) Crosslink site density plot for iCLIP cDNA libraries after alignment to the human genome and random barcode evaluation. ( B) Analysis of FASTKD2-GFP immunoprecipitates by polyacrylamide gel electrophoresis and silver stain. The asterisk denotes a lower molecular mass species of unknown identity. ( A) Titration of tetracycline concentrations required for equal expression of endogenous FASTKD2 and FASTKD2-GFP. Identification of FASTKD2 interacting transcripts in HEK293 cells. Published by Cold Spring Harbor Laboratory Press for the RNA Society. Mendelian disease RNA-binding proteins iCLIP mitochondria oxidative phosphorylation transcript processing. This work identifies key aspects of the molecular network of a previously uncharacterized, disease-relevant RNA-binding protein, FASTKD2, by a combination of genomic, molecular, and metabolic analyses. Metabolic phenotyping of FASTKD2-deficient cells reveals impaired cellular respiration with reduced activities of all respiratory complexes. CRISPR-mediated deletion of FASTKD2 leads to aberrant processing and expression of RNR2 and ND6 mRNA that encodes a subunit of the respiratory complex I. FASTKD2 interacts with a defined set of mitochondrial transcripts including 16S ribosomal RNA (RNR2) and NADH dehydrogenase subunit 6 (ND6) messenger RNA. To investigate whether RNA binding of FASTKD2 contributes to the disease phenotype, we identified the RNA targets of FASTKD2 by iCLIP. A mutation within one of these newly assigned FASTK RBPs, FASTKD2, causes a rare form of Mendelian mitochondrial encephalomyopathy. RNA interactome capture identified several disease-relevant RNA-binding proteins (RBPs) with noncanonical RNA-binding architectures, including all six members of the FASTK (FAS-activated serine/threonine kinase) family of proteins. Mitochondrial RNA processing is an essential step for the synthesis of the components of the electron transport chain in all eukaryotic organisms, yet several aspects of mitochondrial RNA biogenesis and regulation are not sufficiently understood. ![]()
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