Crystal structure of human DGCR8 core

A complex of Drosha with DGCR8 (or its homolog Pasha) cleaves primary microRNA (pri-miRNA) substrates into precursor miRNA and initiates the microRNA maturation process. Drosha provides the catalytic site for this cleavage, whereas DGCR8 or Pasha provides a frame for anchoring substrate pri-miRNAs. To clarify the molecular basis underlying recognition of pri-miRNA by DGCR8 and Pasha, we determined the crystal structure of the human DGCR8 core (DGCR8S, residues 493-720). In the structure, the two double-stranded RNA-binding domains (dsRBDs) are arranged with pseudo two-fold symmetry and are tightly packed against the C-terminal helix. The H2 helix in each dsRBD is important for recognition of pri-miRNA substrates. This structure, together with fluorescent resonance energy transfer and mutational analyses, suggests that the DGCR8 core recognizes pri-miRNA in two possible orientations. We propose a model for DGCR8's recognition of pri-miRNA.     

SUMOylation at K707 of DGCR8 controls direct function of primary microRNA

DGCR8 (DiGeorge syndrome critical region gene 8) is essential for primary microRNA (pri-miRNA) processing in the cell nucleus. It specifically combines with Drosha, a nuclear RNase III enzyme, to form the Microprocessor complex (MC) that cleaves pri-miRNA to precursor miRNA (pre-miRNA), which is further processed to mature miRNA by Dicer, a cytoplasmic RNase III enzyme. Increasing evidences suggest that pri-/pre-miRNAs have direct functions in regulation of gene expression, however the underlying mechanism how it is fine-tuned remains unclear. Here we find that DGCR8 is modified by SUMO1 at the major site K⁷⁰⁷, which can be promoted by its ERK-activated phosphorylation. SUMOylation of DGCR8 enhances the protein stability by preventing the degradation via the ubiquitin proteasome pathway. More importantly, SUMOylation of DGCR8 does not alter its association with Drosha, the MC activity and miRNA biogenesis, but rather influences its affinity with pri-miRNAs. This altered affinity of DGCR8 with pri-miRNAs seems to control the direct functions of pri-miRNAs in recognition and repression of the target mRNAs, which is evidently linked to the DGCR8 function in regulation of tumorigenesis and cell migration. Collectively, our data suggest a novel mechanism that SUMOylation of DGCR8 controls direct functions of pri-miRNAs in gene silencing.     

MD Simulations of the dsRBP DGCR8 Reveal Correlated Motions that May Aid pri-miRNA Binding

Over the past decade, microRNAs (miRNAs) have been shown to affect gene regulation by basepairing with messenger RNA, and their misregulation has been directly linked with cancer. DGCR8, a protein that contains two dsRNA-binding domains (dsRBDs) in tandem, is vital for nuclear maturation of primary miRNAs (pri-miRNAs) in connection with the RNase III enzyme Drosha. The crystal structure of the DGCR8 Core (493-720) shows a unique, well-ordered structure of the linker region between the two dsRBDs that differs from the flexible linker connecting the two dsRBDs in the antiviral response protein, PKR. To better understand the interfacial interactions between the two dsRBDs, we ran extensive MD simulations of isolated dsRBDs (505-583 and 614-691) and the Core. The simulations reveal correlated reorientations of the two domains relative to one another, with the well-ordered linker and C-terminus serving as a pivot. The results demonstrate that motions at the domain interface dynamically impact the conformation of the RNA-binding surface and may provide an adaptive separation distance that is necessary to allow interactions with a variety of different pri-miRNAs with heterogeneous structures. These results thus provide an entry point for further in vitro studies of the potentially unique RNA-binding mode of DGCR8.     

A central role for the primary microRNA stem in guiding the position and efficiency of Drosha processing of a viral pri-miRNA

Processing of primary microRNA (pri-miRNA) stem–loops by the Drosha–DGCR8 complex is the initial step in miRNA maturation and crucial for miRNA function. Nonetheless, the underlying mechanism that determines the Drosha cleavage site of pri-miRNAs has remained unclear. Two prevalent but seemingly conflicting models propose that Drosha–DGCR8 anchors to and directs cleavage a fixed distance from either the basal single-stranded (ssRNA) or the terminal loop. However, recent studies suggest that the basal ssRNA and/or the terminal loop may influence the Drosha cleavage site dependent upon the sequence/structure of individual pri-miRNAs. Here, using a panel of closely related pri-miRNA variants, we further examine the role of pri-miRNA structures on Drosha cleavage site selection in cells. Our data reveal that both the basal ssRNA and terminal loop influence the Drosha cleavage site within three pri-miRNAs, the Simian Virus 40 (SV40) pri-miRNA, pri-miR-30a, and pri-miR-16. In addition to the flanking ssRNA regions, we show that an internal loop within the SV40 pri-miRNA stem strongly influences Drosha cleavage position and efficiency. We further demonstrate that the positions of the internal loop, basal ssRNA, and the terminal loop of the SV40 pri-miRNA cooperatively coordinate Drosha cleavage position and efficiency. Based on these observations, we propose that the pri-miRNA stem, defined by internal and flanking structural elements, guides the binding position of Drosha–DGCR8, which consequently determines the cleavage site. This study provides mechanistic insight into pri-miRNA processing in cells that has numerous biological implications and will assist in refining Drosha-dependent shRNA design.     

Cytoplasmic Drosha activity generated by alternative splicing

RNase III enzyme Drosha interacts with DGCR8 to form the Microprocessor, initiating canonical microRNA (miRNA) maturation in the nucleus. Here, we re-evaluated where Drosha functions in cells using Drosha and/or DGCR8 knock out (KO) cells and cleavage reporters. Interestingly, a truncated Drosha mutant located exclusively in the cytoplasm cleaved pri-miRNA effectively in a DGCR8-dependent manner. In addition, we demonstrated that in vitro generated pri-miRNAs when transfected into cells could be processed to mature miRNAs in the cytoplasm. These results indicate the existence of cytoplasmic Drosha (c-Drosha) activity. Although a subset of endogenous pri-miRNAs become enriched in the cytoplasm of Drosha KO cells, it remains unclear whether pri-miRNA processing is the main function of c-Drosha. We identified two novel in-frame Drosha isoforms generated by alternative splicing in both HEK293T and HeLa cells. One isoform loses the putative nuclear localization signal, generating c-Drosha. Further analysis indicated that the c-Drosha isoform is abundant in multiple cell lines, dramatically variable among different human tissues and upregulated in multiple tumors, suggesting that c-Drosha plays a unique role in gene regulation. Our results reveal a new layer of regulation on the miRNA pathway and provide novel insights into the ever-evolving functions of Drosha.     

Dynamic origins of differential RNA binding function in two dsRBDs from the miRNA "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 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 (K(d) = 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.     

Deformability in the cleavage site of primary microRNA is not sensed by the double‐stranded RNA binding domains in the microprocessor component DGCR8

The prevalence of double‐stranded RNA (dsRNA) in eukaryotic cells has only recently been appreciated. Of interest here, RNA silencing begins with dsRNA substrates that are bound by the dsRNA‐binding domains (dsRBDs) of their processing proteins. Specifically, processing of microRNA (miRNA) in the nucleus minimally requires the enzyme Drosha and its dsRBD‐containing cofactor protein, DGCR8. The smallest recombinant construct of DGCR8 that is sufficient for in vitro dsRNA binding, referred to as DGCR8‐Core, consists of its two dsRBDs and a C‐terminal tail. As dsRBDs rarely recognize the nucleotide sequence of dsRNA, it is reasonable to hypothesize that DGCR8 function is dependent on the recognition of specific structural features in the miRNA precursor. Previously, we demonstrated that noncanonical structural elements that promote RNA flexibility within the stem of miRNA precursors are necessary for efficient in vitro cleavage by reconstituted Microprocessor complexes. Here, we combine gel shift assays with in vitro processing assays to demonstrate that neither the N‐terminal dsRBD of DGCR8 in isolation nor the DGCR8‐Core construct is sensitive to the presence of noncanonical structural elements within the stem of miRNA precursors, or to single‐stranded segments flanking the stem. Extending DGCR8‐Core to include an N‐terminal heme‐binding region does not change our conclusions. Thus, our data suggest that although the DGCR8‐Core region is necessary for dsRNA binding and recruitment to the Microprocessor, it is not sufficient to establish the previously observed connection between RNA flexibility and processing efficiency. Proteins 2015; 83:1165–1179. © 2015 Wiley Periodicals, Inc.     

Backbone 1HN, 13C, and 15N resonance assignments of the tandem RNA-binding domains of human DGCR8

Double-stranded RNA binding domain (dsRBD) containing proteins are critical components of the microRNA (miRNA) pathway, with key roles in small RNA biogenesis, modification, and regulation. DiGeorge Critical Region 8 (DGCR8) is a 773 amino acid, dsRBD-containing protein that was originally identified in humans as a protein encoded in the region of chromosome 22 that is deleted in patients with DiGeorge syndrome. Now, it is realized that DGCR8 complements the nuclear RNase III Drosha to initiate miRNA biogenesis by promoting efficient recognition and cleavage of primary miRNAs (pri-miRNA). A pair of C-terminal tandem dsRBDs separated by a flexible linker are required for pri-miRNA substrate binding and recognition. The crystal structure of the DGCR8 core region comprising residues 493–720 revealed that each dsRBD adopts the canonical αβββα fold. However, several residues located in important flexible regions including the β1-β2-loop implicated in canonical dsRNA recognition are absent in the crystal structure and no RNA-bound structure of DGCR8 has been reported. Here we report the ¹H^N, ¹³C, and ¹⁵N backbone resonance assignments of the 24 kDa, 214 amino acid human DGCR8^core (residues 493–706) by heteronuclear NMR spectroscopy. Our assignments lay the foundation for a detailed solution state characterization of the dynamical and RNA-binding properties of this protein in solution.     

A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting

During microRNA (miRNA) biogenesis, the Microprocessor complex (MC), composed minimally of Drosha, an RNaseIII enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary-miRNA (pri-miRNA) to release the pre-miRNA stem–loop structure. Size-exclusion chromatography of the MC, isolated from mammalian cells, suggested multiple copies of one or both proteins in the complex. However, the exact stoichiometry was unknown. Initial experiments suggested that DGCR8 bound pri-miRNA substrates specifically, and given that Drosha could not be bound or cross-linked to RNA, a sequential model for binding was established in which DGCR8 bound first and recruited Drosha. Therefore, many laboratories have studied DGCR8 binding to RNA in the absence of Drosha and have shown that deletion constructs of DGCR8 can multimerize in the presence of RNA. More recently, it was demonstrated that Drosha can bind pri-miRNA substrates in the absence of DGCR8, casting doubt on the sequential model of binding. In the same study, using a single-molecule photobleaching assay, fluorescent protein-tagged deletion constructs of DGCR8 and Drosha assembled into a heterotrimeric complex on RNA, comprising two DGCR8 molecules and one Drosha molecule. To determine the stoichiometry of Drosha and DGCR8 within the MC in the absence of added RNA, we also used a single-molecule photobleaching assay and confirmed the heterotrimeric model of the human MC. We demonstrate that a heterotrimeric complex is likely preformed in the absence of RNA and exists even when full-length proteins are expressed and purified from human cells, and when hAGT-derived tags are used rather than fluorescent proteins.     

A Highlights from MBoC Selection: Drosha protein levels are translationally regulated during Xenopus oocyte maturation

MicroRNAs (miRNAs) are ∼21-nucleotide-long, single-stranded noncoding RNAs that regulate gene expression. Biogenesis of miRNAs is mediated by the two RNase III-like enzymes, Drosha and Dicer. Here we study miRNA biogenesis during maturation of Xenopus oocytes to eggs using microinjection of pri-miRNAs. We show that processing of exogenous and endogenous primary miRNAs (pri-miRNAs) is strongly enhanced upon maturation of oocytes to eggs. Overexpression of cloned Xenopus Drosha in oocytes, however, boosts pri-miRNA processing dramatically, indicating that Drosha is a rate-limiting factor in Xenopus oocytes. This developmental regulation of Drosha is controlled by poly(A) length addition to the Drosha mRNA, which boosts translation upon transition from oocytes to eggs. Processing of pri-miRNAs by Drosha and Dicer has been shown to be affected by adenosine-to-inosine deamination–type RNA editing. Using activated Xenopus eggs for microinjection experiments, we demonstrate that RNA editing can reduce pri-miRNA processing in vivo. This processing block is determined by the structural but not sequence changes introduced by RNA editing.