Coupling translation termination to RNA surveillance: roles of release factors in quality control and RNA degradation pathways in mammalian mitochondria

Accurate protein synthesis depends on the proper recognition of stop codons by release factors (RFs). The human mitochondrial genome deviates from the standard genetic code by using not only the canonical stop codons UAG and UAA but also two non-canonical stop codons, AGA and AGG. Consequently, human mitochondria employ two release factors—mtRF1a and mtRF1—that recognize canonical and non-canonical stop codons, respectively. Surprisingly, mtRF1 is conserved across vertebrates, despite the absence of non-canonical stop codons in many species. Here, we investigate the function of mtRF1 in Mus musculus, whose mitochondrial open reading frames (mt-ORFs) lack non-canonical stop codons. Using a combination of biochemical approaches and mitoribosome profiling, we demonstrate that mtRF1 retains functional activity as a non-canonical RF, mediating an alternative release mechanism for COX1 via stop codon readthrough. Similarly, in human mitochondria, AGG stop codon readthrough enables ND6 release by mtRF1a in the absence of mtRF1. Our data reveal that mitoribosome stop codon readthrough is a conserved mechanism that compensates for the loss of RFs. While this mechanism facilitates access to alternative stop codons, it is also associated with RNA degradation. A targeted siRNA screen shows that components of the mitochondrial degradosome—including PNPase and SUV3—as well as the mitochondrial poly(A) polymerase MTPAP, participate in this decay pathway. Together, our study provides new insights into mitochondrial translation termination and RNA quality control, revealing stop codon readthrough as a rescue mechanism that enables the completion of mitochondrial protein synthesis when standard termination fails.