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

The first post-transcriptional step in mammalian mitochondrial gene expression, required for the synthesis of the 13 polypeptides encoded in mtDNA, is endonucleolytic cleavage of the primary polycistronic transcripts. Excision of the mtDNA-encoded tRNAs releases most mature RNAs; however, processing of three non-canonical mRNAs not flanked by tRNAs (CO1, CO3, CYB) requires FASTKD5. To investigate the molecular mechanism involved, we created knockout human cell lines to use as assay systems. The absence of FASTKD5 produced a severe OXPHOS assembly defect due to the inability to translate two unprocessed non-canonical mRNAs and predicted altered folding patterns specifically at the 5’-end of the CO1coding sequence. Structural features 13-15 nt upstream of the CO1 and CYB cleavage sites suggest FASTKD5 recognition mechanisms. Remarkably, a map of essential FASTKD5 amino acid residues revealed RNA substrate specificity; however, a key, putative active site residue was required for processing all three non-canonical pre-RNAs. Mutating this site resulted in a substantial increase in binding affinity with all client RNA substrates. A reconstituted in vitro system showed that wild-type, but not mutant, FASTKD5, was able to cleave client substrates correctly. These results establish FASTKD5 as the missing piece of biochemical machinery required to completely process the primary mitochondrial transcript. This study was designed to investigate the structure of human mitochondrial mRNAs. Briefly, mitochondria isolated from HEK-293T cells were incubated with DMS and RNA was extracted from isolated mitochondria. An off the shelf IDT RNAseq library preparation kit was used in conjunction with TGIRT to mediated mutagenesis where DMS modifications occur. This sequencing data was then aligned to the mitochondrial genome and structural models were developed using SEISMIC-RNA (Rouskin Lab) and RNAstructure (Mathews lab).