Overproduction of yeast viruslike particles by strains deficient in a mitochondrial nuclease.

Abstract

Saccharomyces cerevisiae strains are often host to several types of cytoplasmic double-stranded RNA (dsRNA) genomes, some of which are encapsidated by the L-A dsRNA product, an 86,000-dalton coat protein.Here we present the finding that nuclear recessive mutations in the NUCI gene, which encodes the major nonspecific nuclease of yeast mitochondria, resulted in at least a 10-fold increase in amounts of the L-A dsRNA and its encoded coat protein. The effect of nucl mutations on L-A abundance was completely suppressed in strains that also hosted the killer-toxin-encoding M dsRNA. Both NUCI and nuci strains containing the L-A genome exhibited an increase in coat protein abundance and a concomitant increase in L-A dsRNA when the cells were grown on a nonfermentable carbon source rather than on glucose, an effect independent of the increase in coat protein due to nuci mutations or to the absence of M. The increase in L-A expression in nuci strains was similar to that observed in strains with mutations in the nuclear gene encoding the most abundant outer mitochondrial membrane protein, porin. nuci mutations did not affect the level of porin in the mitochondrial outer membrane. Since the effect of mutations in nuci was to alter the copy number of the L-A coat protein genome rather than to change the level of the M toxin genome (as do mak and ski mutations), these mutations define a new class of nuclear genes affecting yeast dsRNA abundance.Most Saccharomyces cerevisiae strains are host to one or more double-stranded RNAs (dsRNAs) (2, 35; for a review, see reference 39). The L-A and M dsRNAs are associated with cytoplasmic virus-like particles and are responsible for conferring the killer phenotype (2, 30). Killer strains secrete a toxin encoded on dsRNAs of the M type (1.8 or 1.5 kilobases [kb]) that can kill sensitive yeast strains (3, 4). The coat proteins that encapsidate the dsRNAs are encoded on the separate L-A genome, which is 4.5 kb in length (1,19,20,31). That host functions are necessary for maintenance and propagation of the dsRNAs is evidenced by the myriad of nuclear genes defined by mutations that eliminate toxin production (MAK [maintenance of killer]) (1,36,40) or enhance the killer phenotype (SKI [superkiller]) (27,33). mak mutations result in the loss of either the toxin-encoding M genome alone or both the L and M genomes, whereas ski mutations result in an increased copy number of the toxinencoding M genome and therefore bestow an enhanced killer phenotype.Here, we present the finding that mutations in a nuclear gene resulted in an increased copy number of the L-A type genome and an overabundance of one product of that genome, the 86-kilodalton (kDa) coat protein. Furthermore, we show that the L-A copy number increased in mutant strains only when M, the toxin-encoding genome, was absent. These nuclear mutations are in the NUCI gene, which encodes the major nonspecific nuclease in yeast mitochondria (5,34,41). We created such a mutation in the course of our studies on CBPI, which is adjacent to NUCI ...

Point mutations upstream of the yeast ADH2 poly(A) site significantly reduce the efficiency of 3'-end formation.

“…However, there may be a weak signal in the negative orientation for this gene, since ,B-galactosidase levels were reduced compared with those generated by pHZ18A2 or pL102. The presence of overlapping polyadenylation signals may be a common feature of convergently transcribed yeast genes (29,42,46,47,51). It is also clear from these results that there is a wide variation in the efficiency at which specific sequences direct 3'-end formation.…”

Yeast CBP1 mRNA 3' end formation is regulated during the induction of mitochondrial function.

“…The first mutant (B2L) has a disruption in the 3' end of an open reading frame (URF, unknown function) which ends 260 bp upstream of the CBPI translational start codon and is transcribed from the same strand as CBPI (36). A second mutant, LNUC (33), has a disruption in the NUCI open reading frame located downstream of CBPI on the opposite strand. NUCI encodes a nonspecific mitochondrial nuclease (64), and the reading frame ends just 244 bp downstream of CBPI.…”

Premature 3′-End Formation of CBP1 mRNA Results in the Downregulation of Cytochrome b mRNA during the Induction of Respiration in Saccharomyces cerevisiae

“…The CBP1 fragment was ligated to the full-length CBP1 gene in plasmid pKim3R in place of the wild-type sequence (which had a BssHII site at ϩ914 to ϩ919 and an NcoI site at ϩ1085 to ϩ1090 added by site-directed mutagenesis). The resulting plasmid (CH-NN/ His) also contains NUC1, which lies immediately 3Ј of CBP1 in the genome (28), interrupted with HIS3 ϩ to allow for selection for integration of the CBP1-NUC1 construct into the genome.…”