Kanako Kuwasako

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.

Summary The pre-spliceosomal complex involves interactions between U1 and U2 snRNPs, where a ubiquitin-like domain (ULD) of SF3A1, a component of U2 snRNP, binds to the stem-loop 4 (SL4; the UUCG tetraloop) of U1 snRNA in U1 snRNP. Here, we reported the 1.80 Å crystal structure of human SF3A1 ULD (ULDSF3A1) complexed with SL4. The structural part of ULDSF3A1 (res. 704–785) adopts a typical β-grasp fold with a topology of β1-β2-α1-310a-β3-β4-310b-β5, closely resembling that of ubiquitin, except for the length and structure of the β1/β2 loop. A patch on the surface formed by three ULDSF3A1-specific residues, Lys756 (β3), Phe763 (β4), and Lys765 (following β4), contacts the canonical UUCG tetraloop structure. In contrast, the directly following C-terminal tail composed of 786KERGGRKK793 was essentially stretched. The main or side chains of all the residues interacted with the major groove of the stem helix; the RGG residues adopted a peculiar conformation for RNA recognition. These findings were confirmed by mutational studies using bio-layer interferometry. Collectively, a unique combination of the β-grasp fold and the C-terminal tail constituting ULDSF3A1 is required for the SL4-specific binding. This interaction mode also suggests that putative post-translational modifications, including ubiquitination in ULDSF3A1, directly inhibit SL4 binding.