MOMEFAST · Molecular Mechanisms Employed by the Newly Assigned RNA-binding Protein FASTKD2

Molecular Mechanisms Employed by the Newly Assigned RNA-binding Protein FASTKD2

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 non-canonical RNA-binding architectures, including all six members of the FASTK (FAS-activated serine/threonine kinase) family of proteins. Mutations within one of these newly assigned FASTK RBPs, FASTKD2, causes a rare form of Mendelian mitochondrial encephalomyopathy. We therefore investigated whether RNA binding of FASTKD2 contributes to the disease phenotype and identified the RNA targets of FASTKD2 by iCLIP (individual nucleotide resolution UV cross-linking and immunoprecipitation). To delineate the mitochondrial target spectrum of FASTKD2, we aligned the FASTKD2-bound RNA target sites with a low false discovery rate to the mitochondrial genome. The resulting peak map revealed the selective association of FASTKD2 with a defined set of mitochondrial transcripts including 16S ribosomal RNA (RNR2) and NADH dehydrogenase subunit 6 (ND6) messenger RNA. We went on to validate these bona fide targets by expression profiling in cell lines depleted of FASTKD2 either by RNA interference or by CRISPR/Cas9-mediated genomic modification. Whereas RNAi-mediated depletion of FASTKD2 mainly affected the processing pattern of the mitochondrial ND6 transcript that encodes a subunit of respiratory complex I (also referred to as NADH dehydrogenase), CRISPR/Cas9-mediated mutagenesis of the FASTKD2 locus in addition lead to aberrant processing and expression of the mitochondrial ribosomal RNA RNR2. Moreover, both modes of depletion of FASTKD2 led to a strong induction of the expression of 7S RNA, a small non-coding RNA encoded in the D-loop region of the mitochondrial genome. This observation is consistent with an induction of mitochondrial transcription, a possible consequence of perturbed mitochondrial homeostasis as 7S RNA levels have previously been proposed to correlate with de novo transcriptional activity in mitochondria. Extensive biochemical and metabolic phenotyping of FASTKD2-deficient cells obtained using the CRISPR/Cas9 system revealed a pronounced global mitochondrial translation defect. As a result FASTKD2 deficiency leads to impaired cellular respiration with reduced activities of all respiratory complexes with subunits encoded on the mitochondrial genome. Taken together, the project at this stage successfully identified 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. The research project “Molecular mechanisms employed by the newly assigned RNA-binding protein FASTKD2” (MoMeFAST) was supported by a Marie Curie Fellowship (FP7/2007-2013 / PIEF-2013-626947).

Data: CORDIS, © European Union