Function in Two dsRBDs from themiRNA “ Microprocessor ” Complex †

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 wasmotivated by electrophoretic mobility shift assays (EMSAs) of the two dsRBDs, which showed that Drosha-dsRBDdoes not bind a representativemiRNAbut isolatedDGCR8-dsRBD1does (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 ofDrosha-dsRBD ismore flexible than the corresponding loop in DGCR8-dsRBD1 but shows no correlation with loop 2, which potentially explains the lack of dsRNA binding byDrosha-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. MicroRNAs (miRNAs) are a large class of gene-regulating RNAs that have been shown to contribute to the control of cellular homeostasis in multicellular organisms by regulating such biologically important processes as apoptosis, cell cycle progression, and cell-type differentiation (1, 2). MaturemiRNAs, roughly 22 nucleotides, regulate gene translation by base pairing with mRNA, and it has been reported that a majority of human genes (>60%) are regulated posttranscriptionally by at least one miRNA (3). Disease states leading to cancer, Alzheimer’s disease, heart disease, and autoimmune diseases have been directly linked to aberrant cellular levels of specific miRNAs (4-8). Maturation of miRNA is a two-step process, with each step being independent and spatially separate. The first step involves cleavage of the single-strandedRNA(ssRNA) tail ofprimarymiRNA(pri-miRNA) in the nucleus by the “microprocessor” complex, which contains theRNase III enzymeDrosha and its cofactor, a double-stranded RNA (dsRNA) binding protein DGCR8 (known as Pasha in Drosophila) (9). After pre-miRNA is transported to the cytosol by exportin-5, the RNase III enzyme Dicer cleaves the terminal loop, thus forming mature miRNA (9). Throughout the maturation of miRNA, dsRNA binding domains (dsRBDs) play vital roles in recruitment and positioning for cleavage (9). dsRBDs are one of the most common RNAbinding motifs, having been reported in viral-encoded proteins and in both prokaryotic and eukaryotic organisms (10). Also, dsRBDs are found in proteins in both the cytoplasm and the nucleus of eukaryotes, as evidenced by the compartmentalization of the first and second stages of miRNAprocessing in the nucleus and cytosol, respectively (9, 11, 12). The dsRBD is an ∼65-68 amino acid motif that forms an RβββR topology, where the two R-helices lie on one face of three antiparallel β-sheets with the other surface of the sheet being solvent-exposed (13-15). The preferred binding partner of dsRBDs is A-form doublehelix RNA, while dsRBDs show little or no affinity for ssRNA, dsDNA, and DNA-RNA hybrids (16, 17). As few as 11 base pairs of dsRNA (coinciding with one turn of canonical A-form helix) have been shown to interact with a single dsRBD (18). Nonsequence specific recognition of dsRNA by dsRBDs is mediated by conserved positively charged residues and hydrogen bond acceptors and donors in loop 2 (L2) and loop 4 (L4), the N-terminal region of helix 2 (H2), and additional contacts from helix 1 (H1) (13, 19-21). These positions of sequence conservation are highlighted inFigure 1 for each dsRBD to be discussed in depth. The dsRBDbindingmode differs from themode employed by the structurally similar RNA recognition motifs (RRMs), exemplified by the two RRMs of U1A, that bind ssRNA by This work was supported by start-up funds provided to S.A. S. by The Pennsylvania State University *To whom correspondence should be addressed. E-mail: sas76@psu. edu. Phone: 814-865-2318. Fax: 814-865-3314. Abbreviations: miRNA, microRNA; ss, single stranded; ds, double stranded; dsRBDs, dsRNA binding domains; DGCR8, DiGeorge critical region 8; NMR, nuclear magnetic resonance; MD, molecular dynamics; EMSA, electrophoretic mobility shift assay; RRMs, RNA recognition motifs; NOE, nuclear Overhauser effect; PKR, dsRNAactivated protein kinase R; PCR, polymerase chain reaction; IPTG, isopropyl β-D-1-thiogalactopranoside; Ni-NTA, nickel nitrilotriacetic acid; MWCO,molecular weight cutoff; GST, glutathione S-transferase; Tris, tris(hydroxymethyl)aminomethane; HEPES, N-(2-hydroxyethyl)piperazine-N0-2-ethanesulfonic acid; PES, polyether sulfone; NTPs, nucleoside triphosphates; TEN250, 10 mM Tris, 1 mM EDTA, and 250 mMNaCl; HSQC, heteronuclear single-quantum coherence; rmsd, root-mean-square deviation. Article Biochemistry, Vol. 49, No. 50, 201