Project description: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. We report that RNAi is present in other budding-yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate siRNAs, which mostly correspond to transposable elements and YM-BM-4 subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess YM-BM-4 mRNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a novel 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. Employ high-throughput sequencing of endogenous small RNAs from the budding yeasts Saccharomyces castellii, Kluyveromyces polysporus, Candida albicans, Saccharomyces cerevisiae, and Saccharomyces bayanus.
Project description: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. We report that RNAi is present in other budding-yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate siRNAs, which mostly correspond to transposable elements and Y´ subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess Y´ mRNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a novel 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.
Project description:The Dicer ribonuclease III (RNase III) enzymes process long double-stranded RNA (dsRNA) into the small interfering RNAs (siRNAs) that direct RNA interference. Here, we describe the structure and activity of a catalytically active fragment of Kluyveromyces polysporus Dcr1, which represents the noncanonical Dicers found in budding yeast. The crystal structure reveals a homodimer that resembles bacterial RNase III but includes a novel N-terminal domain and newly identified catalytic residues conserved throughout eukaryotic RNase III enzymes. Biochemical analyses show that Dcr1 dimers bind cooperatively along the dsRNA substrate and cleave at precise intervals based on the distance between consecutive active sites. Thus, unlike canonical Dicers, which successively remove siRNA duplexes from the dsRNA termini, Dcr1 initiates processing in the interior and works outward. The distinct mechanism of budding-yeast Dicers establishes a novel paradigm for natural protein-based molecular rulers and imparts substrate preferences with ramifications for biological function. High-throughput sequencing of small RNAs generated by in vitro processing of long dsRNA using Kluyveromyces polysporus Dicer
Project description:The Dicer ribonuclease III (RNase III) enzymes process long double-stranded RNA (dsRNA) into the small interfering RNAs (siRNAs) that direct RNA interference. Here, we describe the structure and activity of a catalytically active fragment of Kluyveromyces polysporus Dcr1, which represents the noncanonical Dicers found in budding yeast. The crystal structure reveals a homodimer that resembles bacterial RNase III but includes a novel N-terminal domain and newly identified catalytic residues conserved throughout eukaryotic RNase III enzymes. Biochemical analyses show that Dcr1 dimers bind cooperatively along the dsRNA substrate and cleave at precise intervals based on the distance between consecutive active sites. Thus, unlike canonical Dicers, which successively remove siRNA duplexes from the dsRNA termini, Dcr1 initiates processing in the interior and works outward. The distinct mechanism of budding-yeast Dicers establishes a novel paradigm for natural protein-based molecular rulers and imparts substrate preferences with ramifications for biological function.