The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo

Abstract

The yeast superkiller (SKI ) genes were originally identified from mutations allowing increased production of killer toxin encoded by M "killer" virus, a satellite of the dsRNA virus L-A. XRN1 (SKI1) encodes a cytoplasmic 59-exoribonuclease responsible for the majority of cytoplasmic RNA turnover, whereas SKI2, SKI3, and SKI8 are required for normal 39-degradation of mRNA and for repression of translation of poly(A) minus RNA. Ski2p is a putative RNA helicase, Ski3p is a tetratricopeptide repeat (TPR) protein, and Ski8p contains five WD-40 (beta-transducin) repeats. An xrn1 mutation in combination with a ski2, ski3, or ski8 mutation is lethal, suggesting redundancy of function. Using functional epitopetagged Ski2, Ski3, and Ski8 proteins, we show that Ski2p, Ski3p, and Ski8p can be coimmunoprecipitated as an apparent heterotrimeric complex. With epitope-tagged Ski2p, there was a 1:1:1 stoichiometry of the proteins in the complex. Ski2p did not associate with Ski3p in the absence of Ski8p, nor did Ski2p associate with Ski8p in the absence of Ski3p. However, the Ski3p/Ski8p interaction did not require Ski2p. In addition, ski6-2 or ski4-1 mutations or deletion of SKI7 did not affect complex formation. The identification of a complex composed of Ski2p, Ski3p, and Ski8p explains previous results showing phenotypic similarity between mutations in SKI2, SKI3, and SKI8. Indirect immunofluorescence of Ski3p and subcellular fractionation of Ski2p and Ski3p suggest that Ski2p and Ski3p are cytoplasmic. These data support the idea that Ski2p, Ski3p, and Ski8p function in the cytoplasm in a 39-mRNA degradation pathway.

The human PAF complex coordinates transcription with events downstream of RNA synthesis

“…These data collectively suggest that hSKI, a protein complex that is required for 3Ј-to-5Ј RNA decay in yeast (Masison et al 1995;Brown et al 2000), associates with transcriptionally active genes via its interaction with the hPAF complex in the case of human cells. Notably, Exosome, the other complex that is required for RNA 3Ј-5Ј decay, was also reported to be associated with transcriptionally active genes via its interaction with the transcription elongation factor Spt6 (Andrulis et al 2002).…”

“…hSki8 is also a component of the human SKI (hSKI) complex. The yeast SKI complex and Exosome are both genetically required for 3Ј-to-5Ј mRNA decay (Ridley et al 1984;Masison et al 1995;Anderson and Parker 1998;Brown et al 2000;Araki et al 2001;Mitchell and Tollervey 2003). We found that hSKI localizes to transcriptionally active genes in an hPAF-dependent manner.…”

“…Yeast Ski8, together with Ski2 and Ski3, form a trimeric complex that is required for 3Ј-to-5Ј mRNA decay (Ridley et al 1984;Masison et al 1995;Brown et al 2000). To further analyze the association of hSki8 with the hPAF complex, we established a stable cell line expressing Flag-hSki8.…”

“…The Exosome localizes to both the cytoplasm and the nucleus in yeast and mammals (Bousquet-Antonelli et al 2000), whereas the SKI complex appears to be cytoplasmic in yeast (Brown et al 2000). We next investigated the cellular localization of the hSKI complex.…”

Genetic interactions between [ PSI + ] and nonstop mRNA decay affect phenotypic variation

“…Ski2p, Ski3p, and Ski8p form a heterotrimeric complex that interacts with Ski7p and is required for nonstop decay (23,34,35). We therefore deleted the genes for each of these proteins from the [PSI ϩ ] strain and analyzed their effect on ade2-1 expression.…”