title: Evolutionarily conserved roles of the dicer helicase domain in regulating RNA interference processing.
abstract: The enzyme Dicer generates 21-25 nucleotide RNAs that target specific mRNAs for silencing during RNA interference and related pathways. Although their active sites and RNA binding regions are functionally conserved, the helicase domains have distinct activities in the context of different Dicer enzymes. To examine the evolutionary origins of Dicer helicase functions, we investigated two related Dicer enzymes from the thermophilic fungus Sporotrichum thermophile. RNA cleavage assays showed that S. thermophile Dicer-1 (StDicer-1) can process hairpin precursor microRNAs, whereas StDicer-2 can only cleave linear double-stranded RNAs. Furthermore, only StDicer-2 possesses robust ATP hydrolytic activity in the presence of double-stranded RNA. Deletion of the StDicer-2 helicase domain increases both StDicer-2 cleavage activity and affinity for hairpin RNA. Notably, both StDicer-1 and StDicer-2 could complement the distantly related yeast Schizosaccharomyces pombe lacking its endogenous Dicer gene but only in their full-length forms, underscoring the importance of the helicase domain. These results suggest an in vivo regulatory function for the helicase domain that may be conserved from fungi to humans.
authors.affiliation: From the Department of Molecular and Cell Biology.
authors.forename: Mary Anne
authors.initials: MA
authors.lastname: Kidwell
authors.affiliation: From the Department of Molecular and Cell Biology.
authors.forename: Jessica M
authors.initials: JM
authors.lastname: Chan
authors.affiliation: From the Department of Molecular and Cell Biology, Howard Hughes Medical Institute, and Department of Chemistry, University of California at Berkeley and the Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 doudna@berkeley.edu.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D005075:Biological Evolution; D017124:Conserved Sequence; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D015398:Signal Transduction; D018411:Spodoptera; D013175:Sporothrix / Q000737:chemistry* / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D007527:Isoenzymes; D035683:MicroRNAs; D012330:RNA, Double-Stranded; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D043244:Ribonuclease III; D004265:DNA Helicases
keywords: Dicer; Fungi; MicroRNA Biogenesis; RNA Helicase; RNA Interference (RNAi); Ribonuclease