Dynamic Origins of Differential RNA Binding Function in Two dsRBDs from the miRNA “Microprocessor” Complex

Abstract

MicroRNAs (miRNAs) affect gene regulation by base pairing with mRNA and contribute to the control of cellular homeostasis. The first step in miRNA maturation is conducted in the nucleus by the “microprocessor” complex made up of an RNase III enzyme, Drosha, that contains one dsRNA binding domain (dsRBD), and DGCR8, that contains two dsRBDs in tandem. The crystal structure of DGCR8-Core (493–720), containing both dsRBDs, and the NMR solution structure of Drosha-dsRBD (1259–1337) have been reported, but the solution dynamics have not been explored for any of these dsRBDs. To better define the mechanism of dsRNA binding and thus the nuclear maturation step of miRNA processing, we report NMR spin relaxation and MD simulations of Drosha-dsRBD (1259–1337) and DGCR8-dsRBD1 (505–583). The study was motivated by electrophoretic mobility shift assays (EMSAs) of the two dsRBDs, which showed that Drosha-dsRBD does not bind a representative miRNA but isolated DGCR8-dsRBD1 does (Kd = 9.4±0.4 µM). Our results show that loop 2 in both dsRBDs is highly dynamic but the pattern of the correlations observed in MD is different for the two proteins. Additionally, the extended loop 1 of Drosha-dsRBD is more flexible than the corresponding loop in DGCR8-dsRBD1 but shows no correlation with loop 2, which potentially explains the lack of dsRNA binding by Drosha-dsRBD in the absence of the RNase III domains. The results presented in this study provide key structural and dynamic features of dsRBDs that contribute to the binding mechanism of these domains to dsRNA.

Engineering double-stranded RNA binding activity into the Drosha double-stranded RNA binding domain results in a loss of microRNA processing function

“…This suggests that protein-protein interactions may be mediated by the extended α1-β1 loop of Drosha-dsRBD too. We have previously suggested that the dynamics of Drosha-dsRBD’s extended α1-β1 loop may in some way impair dsRNA binding by the domain [18]. In contrast, our present work shows convincingly that the loss of non-canonical but conserved Region 3 motif sequence in this dsRBD is responsible for its loss of dsRNA binding activity.…”

“…Our laboratory has characterized the structure of an in vitro transcribed pri-mir-16-1 construct [33] and used this model to establish the dsRNA binding affinity of the dsRBDs from DGCR8 [18, 28], TRBP [34], and Dicer [23], as summarized in Table 2. Our results have proven to be highly consistent with those reported by other laboratories using related pri-miRNA constructs [35, 36].…”

The Functional Cycle of Rnt1p: Five Consecutive Steps of Double-Stranded RNA Processing by a Eukaryotic RNase III

“…Therefore, both Drosha and Dicer function with partner proteins. Drosha forms the microRNA Microprocessor complex with DGCR8 that contains two dsRBDs (Wostenberg et al, 2010). Dicer functions with TRBP and PACT, each of which contains three dsRBDs (Haase et al, 2005; Kok et al, 2007; Lee et al, 2006).…”

The Core Microprocessor Component DiGeorge Syndrome Critical Region 8 (DGCR8) Is a Nonspecific RNA-binding Protein

“…(27). Standard three-dimensional NMR techniques (38) were employed to obtain and confirm backbone assignments for DGCR8 RBD1 (residues 509 -582) (39). Line width and crosspeak intensities of 1 H, 15 N-TROSY experiments at varying protein/RNA ratios were determined using Sparky, assuming Gaussian line shapes (31 31 P) room temperature probe with triple-axis shielded gradients.…”