Mutations in the G-domain of Ski7 cause specific dysfunction in non-stop decay.

Translational guanosine triphosphatases (GTPases) are a protein superfamily involved in various steps of protein synthesis. The elongation factor (EF)1α GTPase subfamily paralogues, such as EF1α, eRF3, and Hbs1 function in translation elongation, termination, and mRNA surveillance, respectively. These factors, in their guanosine triphosphate (GTP)-bound conformations, bind with the tRNA or tRNA-mimicking proteins eRF1 and Pelota (Dom34 in yeast), respectively, to form structurally similar complexes. These complexes enter the ribosomal A site to participate in genetic decoding or ribosomal rescue steps in a GTP hydrolysis-dependent manner. It was recently revealed that the archaeal homologue of EF1α, aEF1α, binds 2 protein tRNA mimics, aRF1 and aPelota, in addition to tRNAs, suggesting a common GTPase-dependent mechanism underlying these processes1,2,3,4 (Supplementary Fig. S1). The EF1α-like paralogue Ski7 was first identified as a member of the super killer (ski) antiviral system that blocks expression of non-poly(A) mRNA in the budding yeast Saccharomyces cerevisiae5,6,7. Ski7 is involved in an alternative yeast pathway of non-stop decay (NSD) for aberrant mRNA surveillance8,9 and cooperates with the Ski complex and the cytoplasmic exosome that facilitates 3′-to-5′ mRNA decay10. Double-mutant strains such as xrn1Δ ski7Δ, dcp1 ski7Δ, and dcp2 ski7Δ, in which 5′-to-3′ exoribonuclease (Xrn1)7 or mRNA 5′-decapping enzyme subunits (Dcp1/Dcp2)11 responsible for common 5′-to-3′ mRNA decay are defective, exhibit synthetic lethality. Because at least the 3′-to-5′ or the 5′-to-3′ mRNA decay pathway is required for yeast survival, these evidences strongly suggest that Ski7 participates in the general 3′-to-5′ decay pathway as well as in the mRNA surveillance system for aberrant mRNA decay. However, the molecular basis for each distinctive mechanism is unclear. Ski7 consists of an N-terminal (Ski7N) and a C-terminal (Ski7C) region. Ski7C shares overall sequence similarity with the translational GTPase EF1α family proteins. Phylogenetic analysis revealed that Hbs112, which is involved in the no-go decay mRNA surveillance system (NGD) as well as NSD, is the closest paralogue of Ski713. One characteristic feature of Ski7C is the substitution of a conserved histidine residue with serine in the G3 motif that is crucial for GTP hydrolysis14. Ski7 interacts with the Ski complex and the exosome via different regions of Ski7N. Analysis of a whole-Ski7C-deletion mutant, which solely expresses Ski7N, revealed that the Ski7N domain is required and sufficient for 3′-to-5′ mRNA decay8,10. The C-terminal domain was speculated to play specific roles in the degradation of nonstop mRNAs8 by analysis of the whole-Ski7C-deletion mutant and a whole-Ski7N-deletion mutant solely expressing Ski7C. Those studies, using N-/C-terminally truncated proteins, gave important clues to the domains’ function. However, single-amino-acid mutants, which would reveal the specific functional domains of Ski7, haven’t been studied yet. Hence, the molecular dissection of the functional domains of the full-length Ski7 protein at the level of amino acid residues has been awaited. Recently, high-resolution X-ray crystal structures of Ski7C complexed with either intact GTP or GDP-Pi were solved15. The structural conformation including the major nucleotide-binding residues of both forms is quite similar to that of GTP-bound forms of other GTPase paralogues, except for some residues involved in interactions with the γ-phosphate of GTP. Furthermore, purified Ski7C did not exhibit GTP hydrolytic activity, even in the presence of 80S ribosome with/without eRF1/Dom34, suggesting the existence of a yet-unknown Ski7 cofactor that facilitates GTP hydrolysis. Although the structural study of Ski7C provided a clear insight into the highly conserved as well as exceptional feature of Ski7 as a member of translational GTPases, it failed to provide any clue for its functional relationships with the Ski7N domain in the full-length Ski7 protein. Therefore, the functional meaning of the conserved C-terminal GTPase-like domain attached to the essential N terminus remains unsolved. In this study, we successfully identified a series of NSD loss-of-function mutations in both Ski7N and Ski7C via systematic mutagenesis and genetic screening. Structural mapping and additional in vivo analyses suggested that Ski7C plays a specific role in mRNA surveillance as a regulatory module through its G-domain function.