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Model of the interaction between the dsRBD and 20 bp of A-form RNA. Only exposed conserved (predominantly positively charged) side chains are shown. These are likely to be part of the RNA recognition surface. The domain is placed so that it could access both backbone strands, across the narrow major groove. RNA colour scheme: red, phosphates; orange, sugars; light blue, bases. The figure was produced with MOLSCRIPT (Kraulis, 1991).
Source publication
The double-stranded RNA binding domain (dsRBD) is a approximately 70 residue motif found in a variety of modular proteins exhibiting diverse functions, yet always in association with dsRNA. We report here the structure of the dsRBD from RNase III, an enzyme present in most, perhaps all, living cells. It is involved in processing transcripts, such a...
Context in source publication
Context 1
... 2 is extruded and quite flexible in solution but would then be stabilized by an induced fit to the RNA upon binding. Figure 6 shows, for comparison, the proposed binding surface of the dsRBD structure oriented towards an A-form RNA duplex. With the domain positioned so that the conserved surface residues around the x2 N-terminus are oriented toward the RNA, it can be seen that the domain provides sufficient ligands to make contacts to both strands, by crossing either the narrow major groove (as shown) or else the wider minor groove. ...
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Ribonuclease Dicer belongs to the family of RNase III endoribonucleases, the enzymes that specifically hydrolyze phosphodiester bonds found in double-stranded regions of RNAs. Dicer enzymes are mostly known for their essential role in the biogenesis of small regulatory RNAs. A typical Dicer-type RNase consists of a helicase domain, a domain of unkn...
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...
RNase III enzymes cleave double stranded (ds)RNA. This is an essential step for regulating the processing of mRNA, rRNA, snoRNA and other small RNAs, including siRNA and miRNA. Arabidopsis thaliana encodes nine RNase III: four DICER-LIKE (DCL) and five RNASE THREE LIKE (RTL). To better understand the molecular functions of RNase III in plants we de...
BACKGROUND
Dicer is an endonuclease that belongs to the RNase III family and can specifically recognize and cleave double‐stranded RNA (dsRNA). In most insects, there are two Dicer genes, Dicer‐1 (Dcr‐1) and Dicer‐2 (Dcr‐2), which are involved in the micro‐RNA and small‐interfering RNA pathways in many species, respectively. The function of Dicer i...
Drosha is a nuclear RNase III enzyme that initiates processing of regulatory microRNA. Together with partner protein DiGeorge syndrome critical region 8 (DGCR8), it forms the Microprocessor complex, which cleaves precursor transcripts called primary microRNA to produce hairpin precursor microRNA. In addition to two RNase III catalytic domains, Dros...
Citations
... The DGCR8 molecules also contain dsRBDs. The dsRBDs of Drosha and DGCR8 are essential for the recognition and binding of pri-miRNAs, through their conserved αβββα motifs (Kharrat et al., 1995). Specific secondary structures characteristic of this motif, including the N-terminal α-helix, loop between β-strand one and 2, and the C-terminal α-helix recognize features of RNA, such as minor and major grooves and the 2′OH group of the ribose in RNA (Ryter and Schultz, 1998;Partin et al., 2020). ...
MicroRNAs (miRNAs) are small non-coding RNAs that silence gene expression through their interaction with complementary sequences in the 3′ untranslated regions (UTR) of target mRNAs. miRNAs undergo a series of steps during their processing and maturation, which are tightly regulated to fine-tune their abundance and ability to function in post-transcriptional gene silencing. miRNA biogenesis typically involves core catalytic proteins, namely, Drosha and Dicer, and several other RNA-binding proteins (RBPs) that recognize and interact with miRNA precursors and/or their intermediates, and mature miRNAs along with their interacting proteins. The series of RNA-protein and protein-protein interactions are critical to maintaining miRNA expression levels and their function, underlying a variety of cellular processes. Throughout this article, we review RBPs that play a role in miRNA biogenesis and focus on their association with components of the miRNA pathway with functional consequences in the processing and generation of mature miRNAs.
... ; https://doi.org/10.1101/2023.08.06.552166 doi: bioRxiv preprint dsRBPs interact with highly structured dsRNAs via their double-stranded RNA binding motifs/domains (dsRBMs/dsRBDs). dsRBDs are 65-68 amino acid long domains (9) that contain a highly conserved secondary structure (α 1 -β 1 -β 2 -β 3 -α 2 ), where the two α -helices are packed against an antiparallel β -sheet formed by three β -strands (10,11). All the dsRBDs have three common RNA-binding regions: (1) the middle of helix α 1 (E residue), (2) the N-terminal residues of helix α 2 (KKxAK motif), and (3) the loop between β 1 and β 2 strands (GPxH motif), forming a canonical RNA-binding surface that interacts with the consecutive minor, major, and minor groove of a dsRNA (8,12). ...
TAR RNA binding protein (TRBP) has emerged as a key player in the RNA interference (RNAi) pathway, wherein it binds to different pre-miRNAs and siRNAs, each varying in sequence and/or structure. We hypothesize that TRBP displays dynamic adaptability to accommodate heterogeneity in target RNA structures. Thus, it is crucial to ascertain the role of intrinsic and RNA-induced protein dynamics in RNA recognition and binding. We have previously elucidated the role of intrinsic and RNA-induced conformational exchange in the double-stranded RNA-binding domain 1 (dsRBD1) of TRBP in shape-dependent RNA recognition. The current study delves into the intrinsic and RNA-induced conformational dynamics of the TRBP-dsRBD2 and then compares it with the dsRBD1 study carried out previously. Remarkably, the two domains exhibit differential binding affinity to a 12 bp dsRNA owing to the presence of critical residues and structural plasticity. Further, we report that dsRBD2 depicts constrained conformational plasticity when compared to dsRBD1. Although, in the presence of RNA, dsRBD2 undergoes induced conformational exchange within the designated RNA-binding regions and other residues, the amplitude of the motions remains modest when compared to those observed in dsRBD1. We propose a dynamics-driven model of the two tandem domains of TRBP, substantiating their contributions to the versatility of dsRNA recognition and binding.
Significance Statement
Exploring the intricacies of RNA-protein interactions by delving into dynamics-based measurements not only adds valuable insights into the mechanics of RNA-protein interactions but also underscores the significance of conformational dynamics in dictating the functional outcome in such tightly regulated biological processes. In this study, we measure intrinsic and RNA-induced conformational dynamics in the second dsRBD, i.e., TRBP-dsRBD2, and compare the same with that carried out in the first dsRBD (TRBP-dsRBD1) of TRBP protein, a key player of the RNAi pathway. The study unveils the differential conformational space accessible to the two domains of TRBP, even though they both adopt a canonical dsRBD fold, thereby affecting how they interact with target RNAs.
... In previous structural studies, the isolated dsRBDs were shown to bind dsRNA with little or no specificity (Green & Mathews, 1992;St Johnston et al, 1992;Bass et al, 1994;Bycroft et al, 1995;Kharrat et al, 1995;Nanduri et al, 1998;Ryter & Schultz, 1998;Lehmann & Bass, 1999;Fierro-Monti & Mathews, 2000;Ramos et al, 2000;Blaszczyk et al, 2004;Gan et al, 2006;Stefl et al, 2006;Masliah et al, 2018), but several studies have reported that they can bind preferentially when irregularities (e.g., mismatches, internal loops, and stem-loop junctions) are present in the RNA double helix (Stefl et al, 2005(Stefl et al, , 2010Wang et al, 2011;Jayachandran et al, 2016;Lazzaretti et al, 2018;Yadav et al, 2020). It was shown that the irregularities widen the minor groove of the RNA double helix, thereby facilitating the positioning of the a1 helix of the dsRBD on dsRNA. ...
RNase III Dicer produces small RNAs guiding sequence-specific regulations, with important biological roles in eukaryotes. Major Dicer-dependent mechanisms are RNA interference (RNAi) and microRNA (miRNA) pathways, which employ distinct types of small RNAs. Small interfering RNAs (siRNAs) for RNAi are produced by Dicer from long double-stranded RNA (dsRNA) as a pool of different small RNAs. In contrast, miRNAs have specific sequences because they are precisely cleaved out from small hairpin precursors. Some Dicer homologs efficiently generate both, siRNAs and miRNAs, while others are adapted for biogenesis of one small RNA type. Here, we review the wealth of recent structural analyses of animal and plant Dicers, which have revealed how different domains and their adaptations contribute to substrate recognition and cleavage in different organisms and pathways. These data imply that siRNA generation was Dicer's ancestral role and that miRNA biogenesis relies on derived features. While the key element of functional divergence is a RIG-I-like helicase domain, Dicer-mediated small RNA biogenesis also documents the impressive functional versatility of the dsRNA-binding domain.
... Ideally, we wanted to avoid larger domains to prevent a potential occlusion of the catalytic center of ligase 10C. However, many double stranded RNA-binding domains are either of similar or larger size compared to ligase 10C, which led us to select a binding domain from RNase III (rnc) (32). Finally, we also chose an engrailed homeodomain (en) that contains the highly prevalent and evolutionary conserved DNA-binding helix-turnhelix motif (33). ...
... These choices enabled us to compare the performance of different types of binding domains on the catalytic efficiency of ligase 10C. The double-stranded RNA-binding domain contained in rnc from E. coli was selected because it can interact specifically with the RNA minor and major groove of the A-form helix adopted by double stranded RNA and an RNA-DNA duplex (32). Zinc fingers motifs constitute one of the largest protein superfamilies. ...
The function of most proteins is accomplished through the interplay of two or more protein domains and fine-tuned by natural evolution. In contrast, artificial enzymes have often been engineered from a single domain scaffold and frequently have lower catalytic activity than natural enzymes. We previously generated an artificial enzyme that catalyzed an RNA ligation by >2 million-fold but was likely limited in its activity by low substrate affinity. Inspired by nature's concept of domain fusion, we fused the artificial enzyme to a series of protein domains known to bind nucleic acids with the goal of improving its catalytic activity. The effect of the fused domains on catalytic activity varied greatly, yielding severalfold increases but also reductions caused by domains that previously enhanced nucleic acid binding in other protein engineering projects. The combination of the two better performing binding domains improved the activity of the parental ligase by more than an order of magnitude. These results demonstrate for the first time that nature's successful evolutionary mechanism of domain fusion can also improve an unevolved primordial-like protein whose structure and function had just been created in the test tube. The generation of multi-domain proteins might therefore be an ancient evolutionary process.
... These structures revealed that the RIIID is composed of seven α-helices and adopts a unique fold while the dsRBD adopts an α-β-β-β-α topology, as found in other dsRBD containing enzymes [39]. While no complete structure of Ec-RNase III is currently available, Ec-RNase III dsRBD was solved by NMR spectroscopy showing a typical α-β-β-β-α topology [40]. Furthermore, structure prediction performed recently through the AlphaFold program (https://alphafold.ebi.ac.uk, accessed on the 11 October 2021) [41,42] supports a common structure between Ec-RNase III (P0A7Y0) and previously obtained RNase III structures in bacteria. ...
The ribosome is the universal catalyst for protein synthesis. Despite extensive studies, the diversity of structures and functions of this ribonucleoprotein is yet to be fully understood. Deciphering the biogenesis of the ribosome in a step-by-step manner revealed that this complexity is achieved through a plethora of effectors involved in the maturation and assembly of ribosomal RNAs and proteins. Conserved from bacteria to eukaryotes, double-stranded specific RNase III enzymes play a large role in the regulation of gene expression and the processing of ribosomal RNAs. In this review, we describe the canonical role of RNase III in the biogenesis of the ribosome comparing conserved and unique features from bacteria to eukaryotes. Furthermore, we report additional roles in ribosome biogenesis re-enforcing the importance of RNase III.
... These structures revealed that the RIIID is composed of seven α-helices and adopts a unique fold while the dsRBD adopts an α-β-β-β-α topology, as found in other dsRBD containing enzymes [39]. While no complete structure of Ec-RNase III is currently available, Ec-RNase III dsRBD was solved by NMR spectroscopy showing a typical α-β-β-β-α topology [40]. Furthermore, structure prediction performed recently through the AlphaFold program (https://alphafold.ebi.ac.uk, accessed on the 11 October 2021) [41,42] supports a common structure between Ec-RNase III (P0A7Y0) and previously obtained RNase III structures in bacteria. ...
The ribosome is the universal catalyst for protein synthesis. Despite extensive studies,
the diversity of structures and functions of this ribonucleoprotein is yet to be fully understood.
Deciphering the biogenesis of the ribosome in a step-by-step manner revealed that this complexity is
achieved through a plethora of effectors involved in the maturation and assembly of ribosomal RNAs
and proteins. Conserved from bacteria to eukaryotes, double-stranded specific RNase III enzymes
play a large role in the regulation of gene expression and the processing of ribosomal RNAs. In this
review, we describe the canonical role of RNase III in the biogenesis of the ribosome comparing
conserved and unique features from bacteria to eukaryotes. Furthermore, we report additional roles
in ribosome biogenesis re-enforcing the importance of RNase III.
... The dsRBDs of Drosha and DGCR8 contribute to pri-miRNA binding (Figure 2b). The dsRBD binds to dsRNA via a highly conserved αβββα motif [49,50]. Structural analysis indicates that dsRNA is recognized by three regions of the αβββα motif: the N-terminal α-helix 1 (α-1) (region 1); the loop region between β-strand 1 (β-1) and β-strand 2 (β-2) (region 2), which interact with different minor grooves of the dsRNA; and the C-terminal α-helix 2 (α-2) (region 3), which interacts with a major groove between the two minor grooves [51,52] (Figure 3). ...
... The dsRBDs are divided into two subclasses, type-A and type-B. Type-A has a conserved αβββα motif with a high affinity to dsRNA [49,50]. Type-B, also termed half dsRBD, has poorly conserved N-terminal sequences in the αβββα motif and is associated with protein-protein interactions [83,84]. ...
MicroRNAs (miRNAs) are small non-coding RNAs that are about 22 nucleotides in length. They regulate gene expression post-transcriptionally by guiding the effector protein Argonaute to its target mRNA in a sequence-dependent manner, causing the translational repression and destabilization of the target mRNAs. Both Drosha and Dicer, members of the RNase III family proteins, are essential components in the canonical miRNA biogenesis pathway. miRNA is transcribed into primary-miRNA (pri-miRNA) from genomic DNA. Drosha then cleaves the flanking regions of pri-miRNA into precursor-miRNA (pre-miRNA), while Dicer cleaves the loop region of the pre-miRNA to form a miRNA duplex. Although the role of Drosha and Dicer in miRNA maturation is well known, the modulation processes that are important for regulating the downstream gene network are not fully understood. In this review, we summarized and discussed current reports on miRNA biogenesis caused by Drosha and Dicer. We also discussed the modulation mechanisms regulated by double-stranded RNA binding proteins (dsRBPs) and the function and substrate specificity of dsRBPs, including the TAR RNA binding protein (TRBP) and the adenosine deaminase acting on RNA (ADAR).
... The amino acid sequence analysis revealed that CSR3 (228 residues) is rather similar in size to RNase III from E. coli (EcR3), A. aeolicus (AaR3), and T. maritima (TmR3), comprised of 226, 221, and 240 residues, respectively ( Fig. 2A). These three proteins are all prototypical class 1 RNase III enzymes that have been well studied both structurally and functionally (13,19,20). I-TASSER identified the template's structure by using the LOMETS server and then selected and scored the templates with the highest significance in the threading alignments, which were used to simulate a pool of protein structure decoys. ...
Sweet potato virus disease (SPVD), caused by synergistic infection of Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV), is responsible for substantial yield loss all over the world. However, there are currently no approved treatments for this severe disease. The crucial role played by RNase III of SPCSV (CSR3) as RNA silencing suppressor during the viruses' synergistic interaction in sweetpotato makes it an ideal drug target for developing antiviral treatment. In this study, high-throughput screening (HTS) of small molecular libraries targeting CSR3 was initiated by a virtual screen using Glide-docking, allowing the selection of 6,400 compounds out of 136,353. We subsequently developed and carried out a kinetic-based HTS using fluorescence resonance energy transfer technology that isolated 112 compounds. These compounds were validated with dose-response assays including the kinetic-based HTS and binding affinity assays using surface plasmon resonance and microscale thermophoresis. Finally, the interference of the selected compounds with viral accumulation was verified in planta . In summary, we identified five compounds belonging to two structural classes that inhibited CSR3 activity and reduced viral accumulation in plants. These results provide the foundation for developing antiviral agents targeting CSR3 to provide new strategies for controlling sweetpotato virus diseases.
Significance statement
We report here a high-throughput inhibitor identification that targets a severe sweetpotato virus disease caused by co-infection with two viruses (SPCSV and SPFMV). The disease is responsible for up to 90% yield loss. Specifically, we targeted the RNase III enzyme encoded by SPCSV, which plays an important role in suppressing the RNA silencing defense system of sweetpotato plants. Based on virtual screening, laboratory assays, and confirmation in planta , we identified five compounds that could be used to develop antiviral drugs to combat the most severe sweetpotato virus disease.
... DGCR8 has two dsRBDs, so our DGCR8 construct encompassed both dsRBDs [23]. Rnt1, Staufen, and Xlrbpa dsRBDs were selected because they are among the first identified and best-characterized dsRBDs [2,[10][11][12]14,26,27]. DGCR8 and Dicer, like Drosha, are involved in miRNA biogenesis, so we were further interested in determining how a longer L1 in their dsRBDs affects miRNA processing [18,[28][29][30][31]. ...
A large number of proteins involved in RNA metabolism possess a double-stranded RNA-binding domain (dsRBD), whose sequence variations and functional versatilities are still being recognized. All dsRBDs have a similar structural fold: α1-L1-β1-L2-β2-L3-β3-L4-α2 (α represents an α-helix, β a β-sheet, and L a loop conformation between the well-defined secondary structures). Our recent work revealed that the dsRBD in Drosha, which is involved in animal microRNA (miRNA) biogenesis, differs from other dsRBDs by containing a short insertion in its L1 region and that this insertion is important for Drosha function. We asked why the same insertion is excluded in all other dsRBDs and proposed that a longer L1 may be detrimental to their functions. In this study, to test this hypothesis, we inserted the Drosha sequence into several well-known dsRBDs from various organisms. Gel mobility shift assay demonstrated that L1 extension invariably reduced RNA binding by these dsRBDs. In addition, such a mutation in Dicer, another protein involved in miRNA biogenesis, impaired Dicer’s ability to process miRNAs, which led to de-repression of reporter expression, in human cells. Taken together, our results add to the growing appreciation of the diversity in dsRBDs and suggest that dsRBDs have intricate structures and functions that are sensitive to perturbations in the L1 region.
... The interactions between double-stranded RNA (dsRNA) and dsRNA-binding domains (dsRBDs) are involved in complex cellular processes like RNA splicing (Fu et al., 2016), RNA editing (Stephens, Haudenschild and Beal, 2004), RNA maturation (Ha and Kim, 2014), RNA transport across cellular membranes (Wang et al., 2011), etc.; and hence play a central role in regulating and executing major cellular pathways governing life. Structurally, dsRBDs contain α 1 -β 1 -β 2 -β 3 -α 2 fold spanning a length of about 65-70 amino acid (Bycroft et al., 1995;Kharrat et al., 1995;Masliah, Barraud and Allain, 2013). Three regions of the dsRBD namely, 1) middle of helix α 1 , 2) loop between β 1 and β 2 , and 3) N-terminal residues of the helix α 2 are involved in the interactions with the backbone of the dsRNA at its minor-major-minor grove spanning a length of ~12 bp. ...
Double-stranded RNA-binding domains (dsRBDs) are involved in a variety of biological functions via recognition and processing of dsRNAs. Though the primary substrate of the dsRBDs are dsRNAs with A-form helical geometry; they are known to interact with structurally diverse dsRNAs. Here, we have employed two model dsRBDs – TAR-RNA binding protein and Adenosine deaminase that acts on RNA – to understand the role of intrinsic protein dynamics in RNA binding. We have performed a detailed characterization of the residue-level dynamics by NMR spectroscopy for the two dsRBDs. While the dynamics profiles at the ps-ns timescale of the two dsRBDs were found to be different, a striking similarity was observed in the μs-ms timescale dynamics for both the dsRBDs. Motions at fast μs timescale ( k ex > 50000 s ⁻¹ ) were found to be present not only in the RNA-binding residues but also in some allosteric residues of the dsRBDs. We propose that this intrinsic μs timescale dynamics observed independently in two distinct dsRBDs allows them to undergo conformational rearrangement that may aid dsRBDs to target substrate dsRNA from the pool of structurally different RNAs in cellular environment.
Statement of Significance
This study reports for the first time the detailed characterization of microsecond timescale dynamics observed in RNA-binding regions of two distinct double-stranded RNA-binding domains (dsRBDs) using NMR relaxation dispersion experiments. dsRBDs have been known to target topologically distinct dsRNAs. However, the mechanistic details of the structural adaptation of proteins is not fully understood. We propose that the presence of such dynamics may have large-scale implications in understanding the RNA recognition mechanisms by the dsRBDs.
