Microorganisms evolve via mechanisms spanning sexual/parasexual reproduction, mutators, aneuploidy, Hsp90, and even prions. Mechanisms that may seem detrimental can be repurposed to generate diversity. Here we show the human fungal pathogen Mucor circinelloides develops spontaneous resistance to the antifungal drug FK506 (tacrolimus) via two distinct mechanisms. One involves Mendelian mutations that confer stable drug resistance; the other occurs via an epigenetic RNA interference (RNAi)-mediated pathway resulting in unstable drug resistance. The peptidyl-prolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin1. Calcineurin inhibition by FK506 blocks M. circinelloides transition to hyphae and enforces yeast growth2. Mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance (FK506R) and restore hyphal growth. In parallel, RNAi is spontaneously triggered to silence the FKBP12 fkbA gene, giving rise to drug-resistant epimutants. FK506R epimutants readily reverted to the drug-sensitive wild-type (WT) phenotype when grown without drug. The establishment of these epimutants is accompanied by generation of abundant fkbA small RNA (sRNA) and requires the RNAi pathway as well as other factors that constrain or reverse the epimutant state. Silencing involves generation of a double-stranded RNA (dsRNA) trigger intermediate from the fkbA mature mRNA to produce antisense fkbA RNA. This study uncovers a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes.

RNA polymerase II (Pol II) termination is triggered by sequences present in the nascent transcript. Termination of pre-mRNA transcription is coupled to recognition of cis-acting sequences that direct cleavage and polyadenylation of the pre-mRNA. Termination of nonpolyadenylated [non-poly(A)] Pol II transcripts in Saccharomyces cerevisiae requires the RNA-binding proteins Nrd1 and Nab3. We have used a mutational strategy to characterize non-poly(A) termination elements downstream of the SNR13 and SNR47 snoRNA genes. This approach detected two common RNA sequence motifs, GUA[AG] and UCUU. The first motif corresponds to the known Nrd1-binding site, which we have verified here by gel mobility shift assays. We also show that Nab3 protein binds specifically to RNA containing the UCUU motif. Taken together, our data suggest that Nrd1 and Nab3 binding sites play a significant role in defining non-poly(A) terminators. As is the case with poly(A) terminators, there is no strong consensus for non-poly(A) terminators, and the arrangement of Nrd1p and Nab3p binding sites varies considerably. In addition, the organization of these sequences is not strongly conserved among even closely related yeasts. This indicates a large degree of genetic variability. Despite this variability, we were able to use a computational model to show that the binding sites for Nrd1 and Nab3 can identify genes for which transcription termination is mediated by these proteins.RNA polymerases synthesize discrete transcripts by initiating and terminating transcription in response to specific sequence elements. To initiate transcription, DNA sequences direct the binding of transcription initiation factors and polymerase to appropriate sites upstream of genes. Termination signals, providing for the creation of functional 3Ј ends, may reside either in the DNA template or, alternatively, are found in the nascent RNA transcript.Eucaryotic RNA polymerases have evolved distinct mechanisms for termination. RNA polymerase III (Pol III) requires no protein factors but terminates efficiently after transcribing four to six consecutive U residues, presumably due to instability of the RNA-DNA hybrid in the enzyme active site (1, 7). RNA Pol I terminates in response to a protein factor, Reb1, which blocks further elongation by binding to a DNA sequence downstream of the termination site (28). The Reb1 site is situated in such a way that the paused polymerase contains an inherently unstable U-rich RNA-DNA hybrid in the active site.The RNA Pol II termination mechanism is more complex than those employed by the other eucaryotic RNA polymerases, requiring a large multiprotein complex that recognizes the poly(A) signal in the nascent transcript (4,17,20,21,27,43). Deletion or mutation of the poly(A) signal results in a failure to terminate messages at the appropriate site (9, 19). This observation suggests that processing of the nascent message is coupled to termination, but the mechanism of this coupling remains uncertain. In Saccharomyces cerevisiae, mutations in severa...

We carried out a population genomic survey of Saccharomyces cerevisiae diploid isolates and find that many budding yeast strains have high levels of genomic heterozygosity, much of which is likely due to outcrossing. We demonstrate that variation in heterozygosity among strains is correlated with a life-history tradeoff that involves how readily yeast switch from asexual to sexual reproduction under nutrient stress. This trade-off is reflected in a negative relationship between sporulation efficiency and pseudohyphal development and correlates with variation in the expression of RME1, a transcription factor with pleiotropic effects on meiosis and filamentous growth. Selection for alternate lifehistory strategies in natural versus human-associated environments likely contributes to differential maintenance of genomic heterozygosity through its effect on the frequency that yeast lineages experience sexual cycles and hence the opportunity for inbreeding. In addition to elevated levels of heterozygosity, many strains exhibit large genomic regions of loss-of-heterozygosity (LOH), suggesting that mitotic recombination has a significant impact on genetic variation in this species. This study provides new insights into the roles that both outcrossing and mitotic recombination play in shaping the genome architecture of Saccharomyces cerevisiae. This study also provides a unique case where stark differences in the genomic distribution of genetic variation among individuals of the same species can be largely explained by a lifehistory trade-off.T he frequency of sex and the nature of breeding systems have a profound effect on genome variation and evolution. For example, inbred populations have an increased frequency of homozygous genotypes (1), lower effective rates of recombination (2), and smaller effective population sizes relative to outcrossed populations with the same number of individuals (3). Likewise, clonal populations are expected to exhibit high levels of heterozygosity coupled with increased allelic diversity but decreased genotypic diversity relative to sexual populations (4).The budding yeast Saccharomyces cerevisiae is one of the best studied model organisms, but relatively little is known about the importance of sexual versus asexual reproduction and inbreeding versus outcrossing in shaping genome evolution in this species. One recent study estimated that outcrossing occurs approximately once every 50,000 generations in S. cerevisiae (5), but low rates of outcrossing do not preclude the possibility that outcrossing has an important impact on genetic variation. Studies of the closely related yeast Saccharomyces paradoxus suggest that sexual cycles are rare relative to asexual cycles and that when sex does occur it primarily involves inbreeding (6, 7). However, S. paradoxus exhibits distinctly different intra-and interpopulation patterns of variation than does S. cerevisiae (8), and hence these findings may not be generalizable across the Saccharomyces genus.Patterns of heterozygosity are an important indica...