FASTKD2 is an RNA-binding protein required for mitochondrial RNA processing and translation

Abstract

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. RNA interactome capture identified several disease-relevant RNA-binding proteins (RBPs) with noncanonical RNAbinding architectures, including all six members of the FASTK (FAS-activated serine/threonine kinase) family of proteins. A mutation within one of these newly assigned FASTK RBPs, FASTKD2, causes a rare form of Mendelian mitochondrial encephalomyopathy. To investigate whether RNA binding of FASTKD2 contributes to the disease phenotype, we identified the RNA targets of FASTKD2 by iCLIP. FASTKD2 interacts with a defined set of mitochondrial transcripts including 16S ribosomal RNA (RNR2) and NADH dehydrogenase subunit 6 (ND6) messenger RNA. 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. Metabolic phenotyping of FASTKD2-deficient cells reveals impaired cellular respiration with reduced activities of all respiratory complexes. This work identifies key aspects of the molecular network of a previously uncharacterized, disease-relevant RNAbinding protein, FASTKD2, by a combination of genomic, molecular, and metabolic analyses.

A Genome-wide CRISPR Death Screen Identifies Genes Essential for Oxidative Phosphorylation

“…Depletion of any of these genes disrupts mitochondrial translation by decreasing the level of the mitochondrial 16S rRNA. The role of FASTKD2 in regulating mitochondrial RNA metabolism has been corroborated by others (Antonicka and Shoubridge, 2015; Jourdain et al, 2015; Popow et al, 2015), but the absence of FASTKD2 was not sufficient to trigger a glucose/galactose death phenotype in K562 cells. Although at first this result appears to be inconsistent with reports that nonsense mutations in FASTKD2 are responsible for a familial case of mitochondrial encephalomyopathy, it should be noted that patients with FASTKD2 mutations present symptoms that are restricted to the muscle and CNS, and fibroblasts from the same patients are asymptomatic, thus illustrating the tissue specificity of certain mitochondrial diseases (Ghezzi et al, 2008).…”

The process of mammalian mitochondrial protein synthesis

“…RNA immunoprecipitation assays have revealed an association with 16S rRNA, consistent with mTERF3 playing a role in the assembly of the mt-LSU (Wredenberg et al 2013). Other non-mitoribosomal proteins have been shown by RNA immunoprecipitation to bind 16S rRNA, such as a member of the Fas-activated Serine Threonine Kinase family FASTKD2 and the aforementioned helicase DDX28, both of which have been shown to be required for mt-LSU assembly (Antonicka and Shoubridge 2015; Popow et al 2015; Tu and Barrientos 2015). Interestingly, the mAAA-protease, a membrane-spanning homo- or hetero-oligomer of two proteins AFG3L2 and paraplegin, also appears to affect mt-LSU assembly.…”

Insights into the post-transcriptional regulation of the mitochondrial electron transport chain

“…Previously, the FASTK family of FAS-activated serine/threonine kinases has been identified as non-canonical RNA RBPs implicated in mitochondrial physiology [32–34]. FASTKD4 (FAS-activated serine/threonine kinase D4) has been shown to mediate the turnover of a subset of mitochondrially encoded transcripts [35], whereas FASTKD2 (FAS-activated serine/threonine kinase D2) acts as an RBP that interacts with the mitochondrially encoded transcripts 16S ribosomal RNA ( RNR2 ) and the complex I subunit ND6 [36]. Deletion of FASTKD2 results in aberrant processing and expression of both RNR2 and ND6 with a subsequent decrease in the activity of all respiratory complexes, with the exception of CII [36] (Figure 1B).…”

“…FASTKD4 (FAS-activated serine/threonine kinase D4) has been shown to mediate the turnover of a subset of mitochondrially encoded transcripts [35], whereas FASTKD2 (FAS-activated serine/threonine kinase D2) acts as an RBP that interacts with the mitochondrially encoded transcripts 16S ribosomal RNA ( RNR2 ) and the complex I subunit ND6 [36]. Deletion of FASTKD2 results in aberrant processing and expression of both RNR2 and ND6 with a subsequent decrease in the activity of all respiratory complexes, with the exception of CII [36] (Figure 1B). FASTKD2-mediated post-transcriptional regulation of these genes is a critical cellular process because homozygous nonsense mutations in the FASTKD2 gene are associated with mitochondrial encephalomyopathy [33].…”