HMRF1L is a human mitochondrial translation release factor involved in the decoding of the termination codons UAA and UAG

Abstract

While all essential mammalian mitochondrial factors involved in the initiation and elongation phases of translation have been cloned and well characterized, little is known about the factors involved in the termination process. In the present work, we report the functional analysis of human mitochondrial translation release factors (RF). Here, we show that HMRF1, which had been previously denoted as a human mitochondrial RF, was inactive in in vitro translation system, although it is a mitochondrial protein. Instead, we identified another human mitochondrial RF candidate, HMRF1L, and demonstrated that HMRF1L is indeed a mitochondrial protein that functions specifically as an RF for the decoding of mitochondrial UAA and UAG termination codons in vitro. The identification of the functional mitochondrial RF brings us much closer to a detailed understanding of the translational termination process in mammalian mitochondria as well as to the unraveling of the molecular mechanism of diseases caused by the dys‐regulation of translational termination in human mitochondria.

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.…”

“…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. In this respect, measurements of release activity on bacterial ribosomes 11,12 may be less relevant for the activity on the mitoribosome. It should also be mentioned that earlier homology modelling suggested that mtRF1 cannot read any codon but recognizes ribosomes with an empty A-site 32 .…”

“…Previous studies have suggested two different hypotheses for the termination at non-standard stop codons, where the first invokes RNA editing 11 and the second a À 1 frameshift 17 , both leading to the mtRFs effectively reading a UAG codon. Neither of these hypotheses can be completely verified or disproved without an in vitro vertebrate mitochondrial translation assay, but no such system is available at present.…”

“…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

“…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. This protein had greater similarity in sequence and length to each of the two sequence recognition domains present in RF1-rather than RF2-type proteins and was therefore termed mtRF1a.…”