RNAi in budding yeast

Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA.
Science (Impact Factor: 33.61). 10/2009; 326(5952):544-50. DOI: 10.1126/science.1176945
Source: PubMed

ABSTRACT RNA interference (RNAi), a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast Saccharomyces cerevisiae. Here, we show that RNAi is present in other budding yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate small interfering RNAs, which mostly correspond to transposable elements and Y' subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess Y' messenger RNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a previously unknown class of Dicer proteins, bring the tool of RNAi to the study of budding yeasts, and bring the tools of budding yeast to the study of RNAi.

Download full-text


Available from: Gerald Fink, Apr 24, 2014
36 Reads
  • Source
    • "RNAi, a gene-silencing pathway triggered by double-stranded RNA, has been lost in the model budding yeast S. cerevisiae, but the introduction of Dicer (Dcr1) and Argonaute (Ago1) from Saccharomyces castellii leads to the production of artificial siRNAs. RNAi-competent S. cerevisae thus allows the identification of small RNAs (19–23 nts), corresponding to naturally formed RNA duplexes in vivo (Drinnenberg et al., 2009; see Experimental Procedures). We therefore used this RNAi tool to determine the density of small RNAs produced in the presence or absence of Dcr1/Ago1 in both wild-type and xrn1 mutant strains. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Transcriptome analyses have revealed that convergent gene transcription can produce many 3'-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3'-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3'-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3'-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD) in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs) form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5'-3' cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression.
    Cell Reports 09/2015; DOI:10.1016/j.celrep.2015.08.016 · 8.36 Impact Factor
    • "The loss of RNAi in the budding yeast, Saccharomyces cerevisiae (phylum Ascomycota), is believed to result from adaptation to the presence of Saccharomyces cerevisiae L-A virus. This virus and its satellite-encoded 'killer' toxins provide a selective advantage for the host in competition against yeast strains lacking immunity to the toxin (Drinnenberg et al., 2009, 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Analysis of virus-derived small RNAs with high-throughput sequencing has been successful for detecting novel viruses in plants and invertebrates. However, the applicability of this method has not been demonstrated in fungi, although fungi were among the first organisms reported to utilize RNA silencing. Here, we used virus-infected isolates of the fungal species complex Heterobasidion annosum sensu lato as a model system to test whether mycovirus genome segments can be detected with small RNA deep sequencing. Species of Heterobasidion are some of the most devastating forest pathogens in boreal forests. These fungi cause wood decay and are commonly infected with species of Partitiviridae and the yet unassigned virus species Heterobasidion RNA virus 6 (HetRV6). Small RNA deep sequencing allowed the simultaneous detection of all eight double-stranded RNA virus strains known to be present in the tested samples and one putative mitovirus species (family Narnaviridae) with a single-stranded RNA genome, designated here as Heterobasidion mitovirus 1. Prior to this study, no members of the family Narnaviridae had been described as infecting species of Heterobasidion. Quantification of viral double- and single-stranded RNA with quantitative PCR indicated that co-infecting viral species and viruses with segmented genomes can be detected with small RNA deep sequencing despite vast differences in the amount of RNA. This is the first study demonstrating the usefulness of this method for detecting fungal viruses. Moreover, the results suggest that viral genomes are processed into small RNAs by different species of Heterobasidion.
    Journal of General Virology 12/2014; 96(Pt_3). DOI:10.1099/jgv.0.000003 · 3.18 Impact Factor
    • "Plasmid construction pRS406-(AGO1-DCR1) was created by inserting restriction digest fragments containing the coding sequences and associated promoters of AGO1 and DCR1 from pRS404-PTEF-AGO1 and pRS405-PTEF-DCR1 (Drinnenberg et al. 2009) into pRS406. This plasmid was then used to integrate RNAi machinery into our laboratory strains. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Long noncoding RNAs (lncRNAs) are pervasively transcribed across eukaryotic genomes, but functions of only a very small subset of them have been demonstrated. This has led to active debate about whether many of them have any biological functions. In addition, very few regulators of lncRNAs have been identified. We developed a novel genetic screen using reconstituted RNAi in Saccharomyces cerevisiae and systematically identified a large number of putative lncRNA repressors. Among them, we found that four highly conserved chromatin remodeling factors are global lncRNA repressors that play major roles in shaping the eukaryotic lncRNA transcriptome. Importantly, we identified >250 antisense lncRNAs (CRRATs [chromatin remodeling-repressed antisense transcripts]) whose repression by these chromatin remodeling factors is required for the maintenance of normal levels of overlapping mRNA transcripts. Our results strongly suggest that regulation of mRNA through repression of antisense lncRNAs is far more broadly used than previously appreciated.
    Genes & Development 11/2014; 28(21):2348-60. DOI:10.1101/gad.250902.114 · 10.80 Impact Factor
Show more