Exosome-Mediated Recognition and Degradation of mRNAs Lacking a Termination Codon
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Nonstop mRNAs are not detectably deadenylated even when they are stabilized by deletion of SKI7 or several other exosome mutations. One possible explanation is that a stalled ribosome occupies the extreme 3′ end of this mRNA and prevents exonucleases from digesting it. A corollary of this explanation is that in a wild-type strain the exosome or associated proteins can dislodge a stalled ribosome at the 3′ end of the mRNA or initiate 3′-to-5′ decay of the mRNA in the presence of such a ribosome.
To test whether translation of nonstop PGK1 mRNA was required in cis we introduced G 18 in its 5′ UTR. This sequence forms a stable secondary structure and reduces translation by 4 orders of magnitude (27). This reduction in translation severely reduced exosome-mediated decay of the nonstop PGK1 mRNA (half-life = 14 min).
The SKI7 homology with translation factors is most evident in the GTPase domain but multiple sequence alignment shows that the homology extends to the COOH-terminus of Ski7p EF1A and eRF3 (19).
Supplemental data can be found on Science Online at www.sciencemag.org/cgi/content/full/295/5563/2262/DC1.
Alleles encoding either a COOH-terminal truncation or an NH 2 -terminal deletion of Ski7p were integrated into the genome at the SKI7 locus and were expressed from the SKI7 promoter. The NH 2 -terminal deletion removed amino acids 18 through 239 whereas the COOH-terminal truncation removed all amino acids from 265 to the COOH-terminal. The COOH-terminal truncation removes all of the translation factor homology.
Hemagglutinin (HA)–tagged Ski7p was generated as described (28) and introduced into strains that carried a protein A–tagged version of Rrp4p (29) Ski4p or Rrp6p which are subunits of the exosome. As a control we used a similarly HA-tagged version of Ski3p which is known not to copurify with the exosome (10). All five tagged proteins are expressed from their normal genomic locus and are functional (22). Ski3p Ski4p Ski7p and Rrp6 are also expressed from their own promoters whereas Rrp4p is expressed from the GAL10 promoter (29). Protein extracts were prepared by vortexing in the presence of glass beads and 50 mM Tris-HCl (pH 7.5) 50 mM NaCl 2 mM MgCl 2 1 mM β-mercaptoethanol 0.1% NP40 and complete protease inhibitors EDTA free (Roche) and were incubated at 4°C for 1 hour with immunoglobulin G (IgG)–Sepharose beads. The beads were then washed twice with 40 volumes of the extraction buffer and twice with 40 volumes of the extraction buffer containing 1 M NaCl. The proteins bound to the IgG-Sepharose were recovered by boiling in sample buffer.
A. van Hoof P. A. Frischmeyer H. C. Dietz R. Parker data not shown.
We thank the members of the Parker laboratory for insightful comments and A. Sachs for antibodies to Lsm1p. Funded by the Howard Hughes Medical Institute.