Abstract
<jats:p>Mammalian mitochondrial mRNAs (mt-mRNAs) are excised from polycistronic precursors primarily through cleavage of flanking tRNAs. A subset of junctions lacks intervening tRNAs and is processed by a non-canonical mechanism involving members of the FASTK family of RNA-binding proteins; however, the consequences of non-canonical processing for downstream gene expression remain poorly understood. Long-read RNA sequencing of FASTKD4-deficient cells revealed selective processing defects at the ATP8/6-CO3 and ND5-CYB junctions. Although processing of both junctions was impaired, mitoribosome profiling showed that only CYB translational efficiency declined, explaining reduced CYB protein despite unchanged CYB mRNA abundance. Mitoribosomes preferentially initiated on processed CYB transcripts despite the accumulation of unprocessed precursors, but engaged both processed and unprocessed CO3 transcripts. Chemical probing of RNA structure revealed that the unprocessed ND5-CYB junction contains a predicted RNA secondary structure adjacent to the CYB start codon, while the ATP8/6-CO3 junction is unstructured. Our results show that FASTKD4-dependent processing of the ND5-CYB precursor is required for efficient CYB translation and support a model in which non-canonical processing promotes translation of select transcripts by removing precursor RNA structures that hinder mitoribosome engagement.</jats:p>