桑迫 香奈子

Human HBS1L and SKI7 (HBS1LV3) are isoforms encoded by the same gene. HBS1L forms a complex with PELO to recognize ribosomes stalled on non-stop mRNAs and promotes ribosome splitting, whereas SKI7 acts as a bridge between the exosome and the SKI complex to mediate mRNA decay on stalled ribosomes. Despite substantial differences in the sequence and function of their C-terminal regions, the two isoforms share an identical N-terminal domain (termed UBAh) that resembles the ubiquitin binding UBA and CUE domains (collectively referred to as the three-helix bundle ubiquitin-binding [THB–Ub] group). Although UBAh has been predicted to interact with ubiquitin moieties attached to the small subunits of stalled ribosomes, evidence for its interaction with ubiquitin is lacking. Herein, we report the NMR structure of the mouse UBAh–ubiquitin complex. UBAh adopts a three-helix bundle architecture (α1–α2–α3) with unique connecting loops. The hydrophobic patch in UBAh interacts with the Ile44-centered hydrophobic patch of ubiquitin in a binding mode nearly identical to that of the UBA and CUE domains. In contrast, the α1/α2 loop contains a distinctive double β-turn that accommodates the protrusion of the ubiquitin β-turn. The hallmark motif of UBAh, located within and downstream of this loop, was identified as VLGD/E. HSQC titration experiments yielded a dissociation constant of approximately 50 µM for ubiquitin. These findings demonstrate that UBAh specifically interacts with ubiquitin in vitro, providing structural insights into its potential role in recruiting HBS1L–PELO and SKI7 to stalled ribosomes.

The mitochondrial translation system contains two ribosome rescue factors, ICT1 and MTRFR (C12orf65), which hydrolyze peptidyl-tRNA in stalled ribosomes. ICT1 also functions as a ribosomal protein of the mitochondrial large ribosomal subunit (mtLSU) in mice and humans, and its deletion is lethal. In contrast, MTRFR does not share this role. Although loss-of-function mutations in MTRFR have been linked to human mitochondrial diseases, data on this association in other vertebrates are lacking. Here, attempts to generate Mtrfr knockout mice were unsuccessful. However, knockout zebrafish lines were successfully generated for both ict1 and mtrfr (ict1-/- and mtrfr-/-). Both knockout lines appeared healthy and fertile. ict1-/-, mtrfr-/-, and wild-type adult caudal fin cells showed significant differences in mitochondrial morphology. The ict1 deletion affected the network properties more than the number of individuals and networks, whereas the mtrfr deletion exhibited the opposite effect. Additionally, the survival rates of the knockout line larvae were significantly lower than those of the wild-type larvae under starvation conditions. These results suggest that ict1 and mtrfr are required for survival under specific stress conditions, whereas ict1-/- and mtrfr-/- involve different compensatory mechanisms in response to loss of either factor under nonstress conditions. Ict1 proteins from all teleosts, including zebrafish, lack the N-terminal mtLSU-binding motif found in most metazoans, suggesting that Ict1 does not function as a ribosomal protein in teleosts. Thus, Mtrfr may partially compensate for the loss of Ict1. In conclusion, zebrafish appear to exemplify a limited category of vertebrates capable of enduring genetic abnormalities in ict1 or mtrfr.

Escherichia coli possesses three stalled-ribosome rescue factors, tmRNA·SmpB (primary factor), ArfA (alternative factor to tmRNA·SmpB), and ArfB. Here, we examined the susceptibility of rescue factor-deficient strains from E. coli SE15 to various ribosome-targeting antibiotics. Aminoglycosides specifically decreased the growth of the ΔssrA (tmRNA gene) strain, in which the levels of reactive oxygen species were elevated. The decrease in growth of ΔssrA could not be complemented by plasmid-borne expression of arfA, arfB, or ssrAAA to DD mutant gene possessing a proteolysis-resistant tag sequence. These results highlight the significance of tmRNA·SmpB-mediated proteolysis during growth under aminoglycoside stress. In contrast, tetracyclines or amphenicols decreased the growth of the ΔarfA strain despite the presence of tmRNA·SmpB. Quantitative RT-PCR revealed that tetracyclines and amphenicols, but not aminoglycosides, considerably induced mRNA expression of arfA. These findings indicate that tmRNA·SmpB, and ArfA exert differing functions during stalled-ribosome rescue depending on the type of ribosome-targeting antibiotic.