Superkiller mutations in Saccharomyces cerevisiae suppress exclusion of M2 double-stranded RNA by L-A-HN and confer cold sensitivity in the presence of M and L-A-HN.

Abstract

In an mktl host, L-A-HN double-stranded RNA excludes M2 double-stranded RNA at 30°C but not at 20°C. Recessive mutations suppressing the exclusion of M2 by L-A-HN in an mktl host include six ski (superkiller) genes, three of which (ski6, ski7 and ski8) are new genes. The dominant mutations in one gene (MKS50) and recessive mutations in at least two genes (mksl and mks2) suppress M2 exclusion by L-A-HN but do not show other characteristics of ski mutations and thus define a new class of killer-related chromosomal genes. Mutations in ski2, ski3, ski4, ski6, ski7, and ski8 result in increased M copy number at 30°C and prevent the cells from growing at 8°C. Elimination of M double-stranded RNA from a coldsensitive ski-strain results in the loss of cold sensitivity. ski-[KIL-sd1] strains lack L-A-HN, carry L-A-E, and have a lower M1 copy number than do ski- [KIL-kl] strains and are only slightly cold sensitive. The LTS5 (=MAK6) product is required both for low temperature growth and for M1 maintenance or replication. We propose that the elevated levels of M in ski-strains divert the host LTS5 product away from the host and to the M replication process. We also suggest that the essential role of L-A in M replication is protection of M double-stranded RNA from the negative influence of SKI' products.The killer system of Saccharomyces cerevisiae is unique among eucaryotic virus and plasmid systems in the detail in which interactions of viral and host components have been explored and in the large number of host components shown to be involved. This probably reflects the ease with which these aspects can be investigated in S. cerevisiae rather than any difference in the degree to which viral and host functions are intertwined. Numerous examples are known of host restriction of viral growth in mammalian systems, but analysis of the host genes involved is generally not practical. We show here that overproduction of a S. cerevisiae virus, due to a host mutation, results in host pathology, apparently due to over-utilization by the virus of a specific, essential host component.

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

“…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 Ski2, Ski3, and Ski8, all subunits of the SKI complex, are essential for 3Ј-to-5Ј mRNA decay (Ridley et al 1984;Masison et al 1995). Additionally, the ySKI complex interacts with the Exosome, a multi-subunit complex essential for 3Ј-5Ј mRNA decay (Anderson and Parker 1998;Araki et al 2001;Mitchell and Tollervey 2003).…”

“…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.…”

L-A double-stranded RNA viruslike particle replication cycle in Saccharomyces cerevisiae: particle maturation in vitro and effects of mak10 and pet18 mutations.

“…The maklO(Ts) marker was tested by temperature-sensitive maintenance of [HOK] activity, a marker located on L-A dsRNA. The ski2-2 marker was determined by suppression of [NEX] activity in a diploid resulting from a cross with a ski2-2 mktl strain which has M2 and L-A-HN dsRNAs (16). ski2-21ski2-2 mktllmktl homozygous diploids will maintain M2 stably at 30°C in the presence of L-A-HN, while ski2-21+ mktllmktl diploids will lose M2 under these conditions.…”

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

“…The superkiller (SKI ) genes in yeast are encoded in the nuclear genome+ They were initially identified from mutations that caused overexpression of a killer toxin encoded by the endogenous double-stranded RNA, denoted as M, a satellite of the double-stranded RNA virus L-A (Toh-e et al+, 1978;Ridley et al+, 1984;reviewed in Wickner, 1996areviewed in Wickner, , 1996b)+ Subsequent work demonstrated that SKI2, SKI3, SKI6, SKI7, and SKI8 are necessary to repress translation of poly(A) minus RNAs (Widner & Wickner, 1993;Masison et al+, 1995;Benard et al+, 1998Benard et al+, , 1999, whereas SKI1 is XRN1 (Johnson & Kolodner, 1995) and encodes a 59-exoribonuclease (Larimer et al+, 1992)+ In strains lacking both M and L-A, double mutants of xrn1⌬ and either ski2 or ski3 mutations are not viable (Johnson & Kolodner, 1995) or are temperature sensitive (Benard et al+, 1999), depending on strain background+ These observations indicate that the SKI genes are necessary for a general cellular function in addition to an antiviral activity (Johnson & Kolodner, 1995)+ ski8 mutations also are synthetic lethal with xrn1⌬ (Jacobs Anderson & Parker, 1998; J+T+ Brown & A+W+ Johnson, unpubl+ observation)+ More recently, SKI2, SKI3, SKI6, and SKI8 have been shown to be required for a 39-mRNA degradation pathway (Jacobs Anderson & Parker, 1998)+ Ski6p/Rrp41p itself is a 39-exoribonuclease and is an essential component of the exosome (Mitchell et al+, 1997), a large complex of 39-exoribonucleases that is involved in multiple RNA processing events in ribosome biogenesis (Mitchell et al+, 1996(Mitchell et al+, , 1997Zanchin & Goldfarb, 1999)+…”