title: DGCR8 recognizes primary transcripts of microRNAs through highly cooperative binding and formation of higher-order structures.
abstract: DiGeorge critical region 8 (DGCR8) is essential for maturation of microRNAs (miRNAs) in animals. In the cleavage of primary transcripts of miRNAs (pri-miRNAs) by the Drosha nuclease, the DGCR8 protein directly binds and recognizes pri-miRNAs through a mechanism currently controversial. Our previous data suggest that DGCR8 trimerizes upon cooperative binding to pri-mir-30a. However, a separate study proposed a model in which a DGCR8 molecule contacts one or two pri-miRNA molecules using its two double-stranded RNA binding domains. Here, we extensively characterized the interaction between DGCR8 and pri-miRNAs using biochemical and structural methods. First, a strong correlation was observed between the association of DGCR8 with pri-mir-30a and the rate of pri-miRNA processing in vitro. Second, we show that the high binding cooperativity allows DGCR8 to distinguish pri-miRNAs from a nonspecific competitor with subtle differences in dissociation constants. The highly cooperative binding of DGCR8 to a pri-miRNA is mediated by the formation of higher-order structures, most likely a trimer of DGCR8 dimers, on the pri-miRNA. These properties are not limited to its interaction with pri-mir-30a. Furthermore, the amphipathic C-terminal helix of DGCR8 is important both for trimerization of DGCR8 on pri-miRNAs and for the cleavage of pri-miRNAs by Drosha. Finally, our three-dimensional model from electron tomography analysis of the negatively stained DGCR8-pri-mir-30a complex directly supports the trimerization model. Our study provides a molecular basis for recognition of pri-miRNAs by DGCR8. We further propose that the higher-order structures of the DGCR8-pri-miRNA complexes trigger the cleavage of pri-miRNAs by Drosha.
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Faller
authors.forename: Daniel
authors.initials: D
authors.lastname: Toso
authors.forename: Michio
authors.initials: M
authors.lastname: Matsunaga
authors.forename: Ivo
authors.initials: I
authors.lastname: Atanasov
authors.forename: Rachel
authors.initials: R
authors.lastname: Senturia
authors.forename: Yanqiu
authors.initials: Y
authors.lastname: Chen
authors.forename: Z Hong
authors.initials: ZH
authors.lastname: Zhou
authors.forename: Feng
authors.initials: F
authors.lastname: Guo
mesh_terms: D000818:Animals; D053487:DEAD-box RNA Helicases; D004062:DiGeorge Syndrome / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D035683:MicroRNAs; D012330:RNA, Double-Stranded; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases