Abstract
Ski7 functions as a cofactor in both normal mRNA turnover and non-stop mRNA decay (NSD) mRNA surveillance in budding yeast. The N-terminal region of Ski7 (Ski7N) interacts with the ski-complex and the exosome. The C-terminal region of Ski7 (Ski7C) binds guanine nucleotides and shares overall sequence and structural homology with the proteins of the translational GTPase superfamily, especially the tRNA/tRNA-mimic carrier protein subfamilies such as EF1α, eRF3, and Hbs1. Previous reports showed that Ski7N polypeptide functions adequately in vivo, while Ski7C, if any, only slightly. Furthermore, Ski7C does not exhibit GTP-hydrolysing activities under normal conditions. Therefore, the physiological and functional significance of the conserved Ski7C is unclear. Here, we report strong genetic evidence suggesting differential roles for Ski7N and Ski7C in normal and specific mRNA turnover pathways by creating/isolating mutations in both Ski7N and Ski7C conserved motifs using indicator yeast strains. We concluded that Ski7C participates in mRNA surveillance as a regulatory module competitively with the Hbs1/Dom34 complex. Our results provide insights into the molecular regulatory mechanisms underlying mRNA surveillance.
Highlights
Ski[7] consists of an N-terminal (Ski7N) and a C-terminal (Ski7C) region
An assay strain was constructed by integrating a non-stop HIS3 reporter gene construct, which expresses aberrant non-stop mRNA substrate suitable for NSD, into the genomes of a his3∆ ski7∆ strain (BY4727 background)[17] (Fig. 1b)
The dominant-negative effects of the Ski7C mutants as well as that of the WT were nullified in their respective Ski7C-only forms (Supplementary Fig. S10a), while the cellular expression levels of the truncated Ski7C mutants were comparable to that of the wild type (Supplementary Fig. S10b). These results suggested that molecular functions of Ski7C, at least those operated by the Ski7C amino acid residues revealed in this study, are effective absolutely in the presence of Ski7N, i.e. in the full-length form
Summary
Ski[7] consists of an N-terminal (Ski7N) and a C-terminal (Ski7C) region. Ski7C shares overall sequence similarity with the translational GTPase EF1αfamily proteins. 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. The structural study of Ski7C provided a clear insight into the highly conserved as well as exceptional feature of Ski[7] as a member of translational GTPases, it failed to provide any clue for its functional relationships with the Ski7N domain in the full-length Ski[7] protein. 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
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