Translation and M1 double-stranded RNA propagation: MAK18 = RPL41B and cycloheximide curing

Abstract

MAK18 is one of nearly 30 chromosomal genes of Saccharomyces cerevisiae necessary for propagation of the killer toxin-encoding M 1 double-stranded RNA satellite of the L-A double-stranded RNA virus. We have cloned and sequenced MAK18 and find that it is identical to RPL41B, one of the two genes encoding large ribosomal subunit protein L41. The mak18-1 mutant is deficient in 60S subunits, which we suggest results in a preferential decrease in translation of viral poly(A)-deficient mRNA. We have reexamined the curing of M 1 by low concentrations of cycloheximide (G. R. Fink and C. A. Styles, Proc. Natl. Acad. Sci. USA 69:2846-2849, 1972), which is known to act on ribosomal large subunit protein L29. We find that when M 1 is supported by L-A proteins made from the poly(A)؉ mRNA of a cDNA clone of L-A, cycloheximide does not decrease the M 1 copy number, consistent with our hypothesis.Saccharomyces cerevisiae is host to several double-stranded RNA (dsRNA) viruses (reviewed in references 42 and 43). Among them is the L-A virus and its satellite M 1 , a 1.8-kb dsRNA species encapsidated in and replicated by the proteins encoded by L-A's single 4.6-kb dsRNA segment. The viral particles of L-A and M 1 are located in the cytoplasm of the yeast host. L-A and M 1 are very similar to the core particles of dsRNA viruses of higher eukaryotes, reoviruses and rotaviruses, in having replicase and transcriptase activities in the particle and perhaps in their structural symmetry (5).As for the Reoviridae, replication of L-A and M 1 is conservative, with parental strands remaining associated, and sequential, with plus (i.e., coding) and minus strands synthesized at different points of the replication cycle. Viral plus strands are synthesized in the viral particle during a transcription step which uses the minus strand of the parent genome as a template. Newly synthesized plus strands are extruded into the cytoplasm, where they are translated and then encapsidated by viral proteins to form new virus particles. Minus-strand synthesis occurs inside the viral particle during a replication step, and mature viral particles result.The L-A plus strand has two open reading frames (ORFs): ORF1 and ORF2 (16). ORF1 encodes the 76-kDa major coat protein, Gag. A combination of ORF1 and the overlapping ORF2 encodes the 180-kDa Gag-Pol fusion protein formed by a Ϫ1 ribosomal frameshift event (8,14,16). The Pol portion of this fusion protein is involved in the transcription and replication steps of the L-A and M 1 life cycles. The M 1 virus does not encode any proteins needed for its own propagation; however, it does encode a secreted protein toxin and immunity to that toxin (reviewed in reference 4). M 1 is dependent upon the protein products of L-A for its propagation (3, 44).In addition to these two L-A-encoded proteins, propagation of L-A and M 1 is affected by an assortment of chromosomal genes. These genes are divided into two groups: the SKI genes and the MAK genes. The SKI genes repress the copy number of L-A and M 1 and the translation of ...

Decoying the Cap2 mRNA Degradation System by a Double-Stranded RNA Virus and Poly(A)2 mRNA Surveillance by a Yeast Antiviral System

“…The MAK genes were defined by their inability to maintain M 1 dsRNA. Mutations in 20 mak genes lead to diminished levels of free 60S ribosomal subunits (9,40). These mak mutations are suppressed by ski2 mutations (45,57).…”

“…Prompted by our finding that three MAK genes encode proteins of the large ribosomal subunit (MAK8 ϭ L3, MAK7 ϭ L4, MAK18 ϭ L41 [9,40,61]), we have recently found that mutants in 20 of the 27 MAK genes examined have a marked decrease in free 60S ribosomal subunits (9,40). Because the 3Ј poly(A) structure is believed to enhance association of the 60S subunit with the 40S subunit waiting at the initiator AUG (38), a decrease in free 60S subunits should be more detrimental to translation of the poly(A) Ϫ viral mRNAs than to translation of the poly(A) ϩ cellular mRNAs.…”

Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae

“…The killer phenotype is caused by infection of yeast cells by the endogenous L-A and M 1 dsRNA viruses (reviewed in Wickner 1996). The M 1 virus, and hence the killer phenotype, is highly susceptible to defects in the translational apparatus, e.g., to defects in 60S ribosomal subunit biogenesis (Ohtake and Wickner 1995b), to the presence of translational inhibitors (Sommer and Wickner 1982;Carroll and Wickner 1995), to mutations in ribosomal proteins Carroll and Wickner 1995;Ohtake and Wickner 1995a;Meskauskas and Dinman 2001), and to changes in the efficiency of programmed −1 ribosomal frameshifting (−1 PRF; Dinman and Wickner 1992). In order to minimize recombination and select cells containing only mutant forms of 5S rRNA, a rigorous selection scheme involving negative selection for both the wild-type RDN1 gene and the plasmid on which it was encoded was devised (see Materials and methods).…”

Killer toxin of Pichia membranifaciens and its possible use as a biocontrol agent against grey mould disease of grapevine

“…The food and beverage industries were among the first to explore the ability of toxin-producing yeasts to kill other micro-organisms (Javadekar et al, 1995). Attention has mainly focused on the characterization of killer toxins from Saccharomyces cerevisiae (Bevan et al, 1973;Breinig et al, 2002;Carroll & Wickner, 1995;Schmitt & Radler, 1988;Weinstein et al, 1993;Wickner, 1974Wickner, , 1986 and Kluyveromyces lactis (Niwa et al, 1981;Young, 1987), more recently followed by investigation of yeasts such as Zygosaccharomyces bailii (Radler et al, 1993), Hanseniaspora uvarum (Radler et al, 1990), Pichia membranifaciens (Santos et al, 2000), Debaryomyces hansenii (Gunge et al,mycocins are proteins or glycoproteins that bind to polysaccharide structures on the yeast cell wall and this property has been used for the production of purified toxin proteins (Hutchins & Bussey, 1983).…”

“…The food and beverage industries were among the first to explore the ability of toxin-producing yeasts to kill other micro-organisms (Javadekar et al, 1995). Attention has mainly focused on the characterization of killer toxins from Saccharomyces cerevisiae (Bevan et al, 1973;Breinig et al, 2002;Carroll & Wickner, 1995;Schmitt & Radler, 1988;Weinstein et al, 1993;Wickner, 1974Wickner, , 1986 and Kluyveromyces lactis (Niwa et al, 1981;Young, 1987), more recently followed by investigation of yeasts such as Zygosaccharomyces bailii (Radler et al, 1993) mycocins are proteins or glycoproteins that bind to polysaccharide structures on the yeast cell wall and this property has been used for the production of purified toxin proteins (Hutchins & Bussey, 1983).Strains of P. membranifaciens are common contaminants in food-related environments (Heard & Fleet, 1987; Noronha-da-Costa et al, 1995) and occur with high frequency in fermenting olive brines (Marquina et al, 1992(Marquina et al, , 1997. One of the isolates from such an environment, P. membranifaciens CYC 1106, showed a particularly strong, broad-spectrum, zymocidal activity.…”