mtRF1a Is a Human Mitochondrial Translation Release Factor Decoding the Major Termination Codons UAA and UAG

Abstract

SummaryHuman mitochondria contain their own genome, encoding 13 polypeptides that are synthesized within the organelle. The molecular processes that govern and facilitate this mitochondrial translation remain unclear. Many key factors have yet to be characterized—for example, those required for translation termination. All other systems have two classes of release factors that either promote codon-specific hydrolysis of peptidyl-tRNA (class I) or lack specificity but stimulate the dissociation of class I factors from the ribosome (class II). One human mitochondrial protein has been previously identified in silico as a putative member of the class I release factors. Although we could not confirm the function of this factor, we report the identification of a different mitochondrial protein, mtRF1a, that is capable in vitro and in vivo of terminating translation at UAA/UAG codons. Further, mtRF1a depletion in HeLa cells led to compromised growth in galactose and increased production of reactive oxygen species.

Codon-reading specificities of mitochondrial release factors and translation termination at non-standard stop codons

“…Even though the bacterial and the mitochondrial ribosomes are bound to be rather different overall, the fact that the decoding region of the mitochondrial 9S and the bacterial 16S RNA are conserved in secondary structure 4,29 strongly suggests that this also pertains on the local three-dimensional structure. This is also supported by the strong sequence conservation between RF1 and mtRF1a in the decoding region, which, together with the fact that the experimentally observed specificity of mtRF1a on bacterial ribosomes 11,12 , is reproduced here, strengthens the reliability of our derived homology models.…”

“…This could be one reason for the limited effect on protein levels reported on mtRF1a depletion 12 . It is also a distinct possibility that mtRF1 is, in fact, the major release factor in vertebrate mitochondria, as mtRF1 has apparently not been tested in knockdown experiments.…”

“…mtRF1 was identified by bioinformatics and was initially believed to read all four of the mitochondrial stop codons 10 . A new candidate, mtRF1a, was later found and shown to have release activity in vitro on Escherichia coli ribosomes for the UAA and UAG stop codons 11,12 . However, no release activity for mtRF1 was observed at any of the mitochondrial stop codons UAA, UAG, AGA and AGG in these bacterial termination assays.…”

Terminating human mitochondrial protein synthesis: A shift in our thinking

“…Both of the domains involved in sequence recognition, however, are somewhat divergent in sequence and length but this was assumed to allow specificity for this expanded and unusual repertoire of four termination signals in human mitochondria. 10,11 Subsequent biochemical and in vivo analyses, however, failed to reveal any release activity associated with this protein. 12,13 After further bioinformatic mining, a second candidate emerged.…”

“…Both in vitro and in vivo analyses confirmed that mtRF1a was active on UAA and UAG triplets but no activity could be detected with AGA/ AGG codons positioned in the 70S E. coli ribosomal A-site. 11 This accounted for termination of eleven of the thirteen human mt-ORFs. It did not, however, resolve the question of which protein was responsible for release of polypeptides encoded by can recognise all three stop codons.…”