Response to “Ribosome Rescue and Translation Termination at Non-standard Stop Codons by ICT1 in Mammalian Mitochondria”

摘要

Overview In a recent paper published by Akabane et al. [ 1 ], a homologous mitochondrial translation system was supplemented with ICT1, the mitochondrial translation release factor family member. Under these conditions, ICT1 was shown to exhibit general release activity at all codons tested, including AGA/AGG, which are normally redundant in human mitochondria. The authors suggest that this ICT1 activity may occur in vivo, challenging Temperley et al., 2010 [ 2 ]. We wish to point out, however, that the data presented in this paper are in vitro and do not account for the live cell data consistent with a -1 frameshift at AGA/AGG placing a standard UAG stop codon in the human mitoribosomal A-site. Response The impressive data, recently published in Nature and Science , from the groups headed by Ban and by Ramakrishnan have confirmed, through the use of high-resolution cryo-electron microscopy (cryo-EM) and chemical cross-linking–mass spectroscopy, that ICT1 is a component of the mammalian mitochondria [ 3 – 5 ]. ICT1 is a member of a family of release factors, which are ribosome-dependent peptidyl–transfer ribonucleic acid (tRNA) hydrolases. These proteins all contain a highly conserved tripeptide motif, GGQ, that is essential for promoting cleavage of the ester bond that anchors the nascent peptide chain to the resident tRNA [ 6 – 8 ]. In addition to ICT1, these class I release factor (RF) family members include mtRF1a (mtRF1L), mtRF1, and C12orf65. Only one of these, mtRF1a (mtRF1L), has had a clearly defined physiological function ascribed to it. This protein recognises stop codons UAA and UAG to facilitate release activity [ 9 ]. Both ICT1 and C12orf65 are smaller proteins in part due to the loss of the codon recognition domains. Studies to identify their functions, along with those of mtRF1, continue. A little more information is available for ICT1. First principles would suggest that the permanent presence in the ribosome of a protein that can hydrolyse randomly a nascent peptide from the P-site tRNA would be a dangerous design. Experimental evidence has shown that the GGQ motif in ICT1 has retained functionality. Site-directed mutations were introduced in this tripeptide to generate HEK293 cell lines that can express either GSQ or AGQ variants. For each mutant cell line, sucrose gradients and coimmunoprecipitation experiments confirmed incorporation of the variant ICT1 into the mitoribosome. The presence of either version of the mutated ICT1, however, caused growth defects [ 10 ]. The fact that mammalian mitoribosomes have adopted this protein as an integral member of the large subunit but do not suffer from profligate abortive translation events indicates that they have also developed a strategy to control this activity. We were pleased to see the recent article on ICT1 by Akabane et al. that described the use of translationally active mammalian mitoribosomes [ 1 ]. This publication presents in vitro data, which show elegantly that purified ICT1 can release di/tripeptides from mammalian mitoribosomes programmed with a variety of short, synthetic, RNA molecules. The authors suggest this result is in disagreement with work previously published from our laboratory. We believe, however, that the data and conclusions reached by Akabane in their recent publication are in general agreement rather than in contradiction to Richter et al. (2010) [ 10 ]. In our original paper, we observed that free ICT1, in our in vitro assays, was able to demonstrate translational release activity on bacterial ribosomes lacking messenger RNA (mRNA) or any codon, including AGA and AGG (Fig 3C in Richter et al.) [ 10 ]. When ICT1 is in excess and not incorporated into the mitoribosome, it may be able to access the A-site and activate termination of a stalled 55S; it may also be able to do so following slippage of a 55S particle that reached an out-of-frame AGA/AGG or if no mRNA was present in the A-site. The natural state of the cells, however, appears not to retain an excess free pool of ICT1. We performed sucrose gradients to reveal the status of the ribosomal proteins using cells that only contained endogenous levels of ICT1. These show that in HEK293 cells, all detectable protein is integrated into the mitoribosome and that there is negligible, if any, unincorporated ICT1 present (Fig 2E of Richter et al.) [ 10 ]. Furthermore, we were careful to say that uncontrolled activity of ICT1 under normal elongation conditions would be predicted to be detrimental because of the potential to abort elongation. In the absence at that time of any structural information on the position of ICT1 in the mitoribosome, we suggested that ICT1 could display rescue activity only under conditions that cause mitoribosome distortion. We are, therefore, not surprised that no release activity was observed under normal conditions in the in vitro reactions of isolated 55S on substrate in the absence of release factors. The lack of activity of mtRF1a/L a