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Excision of the mtDNA-encoded transfer RNAs (tRNAs) releases most mature RNAs; however, processing of three noncanonical messenger RNAs (mRNAs) not flanked by tRNAs (CO1, CO3, and 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 noncanonical mRNAs and predicted altered folding patterns specifically at the 5\u2032-end of the CO1 coding sequence. Structural features 13\u201315\u00a0nt 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 noncanonical pre-RNAs. Mutating this site did not significantly alter the binding of any client RNA substrate. 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.<\/jats:p>","DOI":"10.1093\/nar\/gkaf665","type":"journal-article","created":{"date-parts":[[2025,6,28]],"date-time":"2025-06-28T07:49:21Z","timestamp":1751096961000},"source":"Crossref","is-referenced-by-count":8,"title":["FASTKD5 processes mitochondrial pre-mRNAs at noncanonical cleavage sites"],"prefix":"10.1093","volume":"53","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2525-2867","authenticated-orcid":false,"given":"Hana","family":"Antonicka","sequence":"first","affiliation":[{"name":"McGill University Department of Human Genetics, , Montreal , QC H3A2B4 ,","place":["Canada"]},{"name":"The Neuro, Montreal Neurological Institute , Montreal , QC H3A2B4 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