title: Drosophila dicer-2 cleavage is mediated by helicase- and dsRNA termini-dependent states that are modulated by Loquacious-PD.
abstract: In previous studies we observed that the helicase domain of Drosophila Dicer-2 (dmDcr-2) governs substrate recognition and cleavage efficiency, and that dsRNA termini are key to this discrimination. We now provide a mechanistic basis for these observations. We show that discrimination of termini occurs during initial binding. Without ATP, dmDcr-2 binds 3' overhanging, but not blunt, termini. By contrast, with ATP, dmDcr-2 binds both types of termini, with highest-affinity binding observed with blunt dsRNA. In the presence of ATP, binding, cleavage, and ATP hydrolysis are optimal with BLT termini compared to 3'ovr termini. Limited proteolysis experiments suggest the optimal reactivity of BLT dsRNA is mediated by a conformational change that is dependent on ATP and the helicase domain. We find that dmDcr-2's partner protein, Loquacious-PD, alters termini dependence, enabling dmDcr-2 to cleave substrates normally refractory to cleavage, such as dsRNA with blocked, structured, or frayed ends.
authors.affiliation: Department of Biochemistry, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Niladri K
authors.initials: NK
authors.lastname: Sinha
authors.affiliation: Department of Biochemistry, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Kyle D
authors.initials: KD
authors.lastname: Trettin
authors.affiliation: Department of Biochemistry, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: P Joseph
authors.initials: PJ
authors.lastname: Aruscavage
authors.affiliation: Department of Biochemistry, University of Utah, Salt Lake City, UT 84112, USA. Electronic address: bbass@biochem.utah.edu.
authors.forename: Brenda L
authors.initials: BL
authors.lastname: Bass
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000737:chemistry / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D006868:Hydrolysis; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D043244:Ribonuclease III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D029721:Drosophila Proteins; C503213:Loqs protein, Drosophila; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D000255:Adenosine Triphosphate; C478069:DCR-2 protein, Drosophila; D043244:Ribonuclease III; D020365:RNA Helicases