FASTKD5 processes mitochondrial pre-mRNAs at non-canonical cleavage sites

The regulation of mammalian mitochondrial gene expression is largely post-transcriptional and the first step in translating the 13 polypeptides encoded in mtDNA is endonucleolytic cleavage of the primary polycistronic transcripts. As the rRNAs and most of the mRNAs in mtDNA are flanked by tRNAs, the release of the mature RNAs occurs mostly by excision of the tRNAs. Processing the non-canonical mRNAs, not flanked by tRNAs, requires FASTKD5, but the molecular mechanism remains unknown. To investigate this, we created and characterized a knockout cell line to use as an assay system. The absence of FASTKD5 resulted in a severe combined OXPHOS assembly defect due to the inability to translate mRNAs with unprocessed 5’-UTRs. Analysis of RNA processing of FASTKD5 variants allowed us to map amino acid residues essential for function. Remarkably, this map was RNA substrate-specific, arguing against a one size fits all model. A reconstituted in vitro system with purified FASTKD5 protein and synthetic RNA substrates showed that FASTKD5 on its own was able to cleave client substrates correctly, but not non-specific RNA sequences. These results establish FASTKD5 as the missing piece of the biochemical machinery required to completely process the primary mitochondrial transcript.