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Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes 1 , 2 . In Drosophila , an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs) 3 , 4 . ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes 5 , 6 . Here we report the cryo-electron microscopy structures of Dcr-2–Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and substantial conformational changes of Dcr-2 during a dsRNA-processing cycle. The N-terminal helicase and domain of unknown function 283 (DUF283) domains undergo conformational changes after initial dsRNA binding, forming an ATP-binding pocket and a 5′-phosphate-binding pocket. The overall conformation of Dcr-2–Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, whereas the interactions between the DUF283 and RIIIDb domains prevent non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active centre. Additional ATP-dependent conformational changes are required to form an active dicing state and precisely cleave the dsRNA into a 21 bp siRNA duplex as confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full cycle of ATP-dependent dsRNA processing by Dcr-2–Loqs-PD. Structures of the Dcr-2–Loqs-PD complex while it is processing a double-stranded RNA (dsRNA) substrate elucidate the interactions between Dcr-2 and Loqs-PD, and show that Dcr-2 undergoes substantial conformational changes during a dsRNA-processing cycle.

In flies, Argonaute2 (Ago2) and small interfering RNA (siRNA) form an RNA-induced silencing complex to repress viral transcripts 1 . The RNase III enzyme Dicer-2 associates with its partner protein R2D2 and cleaves long double-stranded RNAs to produce 21-nucleotide siRNA duplexes, which are then loaded into Ago2 in a defined orientation 2 – 5 . Here we report cryo-electron microscopy structures of the Dicer-2–R2D2 and Dicer-2–R2D2–siRNA complexes. R2D2 interacts with the helicase domain and the central linker of Dicer-2 to inhibit the promiscuous processing of microRNA precursors by Dicer-2. Notably, our structure represents the strand-selection state in the siRNA-loading process, and reveals that R2D2 asymmetrically recognizes the end of the siRNA duplex with the higher base-pairing stability, and the other end is exposed to the solvent and is accessible by Ago2. Our findings explain how R2D2 senses the thermodynamic asymmetry of the siRNA and facilitates the siRNA loading into Ago2 in a defined orientation, thereby determining which strand of the siRNA duplex is used by Ago2 as the guide strand for target silencing. Cryo-electron microscopy structures of Drosophila Dicer-2–R2D2 complexes with and without small interfering RNA reveal how the RNA is presented to Argonaute in the correct orientation for viral gene silencing.

Endogenous RNA transcription characterizes double-stranded RNA (dsRNA) viruses in the Reoviridae, a family that is exemplified by its simple, single-shelled member cytoplasmic polyhedrosis virus (CPV). Because of the lack of in situ structures of the intermediate stages of RNA-dependent RNA polymerase (RdRp) during transcription, it is poorly understood how RdRp detects environmental cues and internal transcriptional states to initiate and coordinate repeated cycles of transcript production inside the capsid. Here, we captured five high-resolution (2.8–3.5 Å) RdRp–RNA in situ structures—representing quiescent, initiation, early elongation, elongation and abortive states—under seven experimental conditions of CPV. We observed the ‘Y’-form initial RNA fork in the initiation state and the complete transcription bubble in the elongation state. These structures reveal that de novo RNA transcription involves three major conformational changes during state transitions. Our results support an ouroboros model for endogenous conservative transcription in dsRNA viruses.

Characterizing the genome of mature virions is pivotal to understanding the highly dynamic processes of virus assembly and infection. Owing to the different cellular fates of DNA and RNA, the life cycles of double-stranded (ds)DNA and dsRNA viruses are dissimilar. In terms of nucleic acid packing, dsDNA viruses, which lack genome segmentation and intra-capsid transcriptional machinery, predominantly display single-spooled genome organizations 1 – 8 . Because the release of dsRNA into the cytoplasm triggers host defence mechanisms 9 , dsRNA viruses retain their genomes within a core particle that contains the enzymes required for RNA replication and transcription 10 – 12 . The genomes of dsRNA viruses vary greatly in the degree of segmentation. In members of the Reoviridae family, genomes consist of 10–12 segments and exhibit a non-spooled arrangement mediated by RNA-dependent RNA polymerases 11 – 14 . However, whether this arrangement is a general feature of dsRNA viruses remains unknown. Here, using cryo-electron microscopy to resolve the dsRNA genome structure of the tri-segmented bacteriophage ɸ6 of the Cystoviridae family, we show that dsRNA viruses can adopt a dsDNA-like single-spooled genome organization. We find that in this group of viruses, RNA-dependent RNA polymerases do not direct genome ordering, and the dsRNA can adopt multiple conformations. We build a model that encompasses 90% of the genome, and use this to quantify variation in the packing density and to characterize the different liquid crystalline geometries that are exhibited by the tightly compacted nucleic acid. Our results demonstrate that the canonical model for the packing of dsDNA can be extended to dsRNA viruses. The cryo-electron microscopy structure of the bacteriophage ɸ6 dsRNA genome shows that the genome is packaged in a spooled manner that is more similar to dsDNA viruses than to other dsRNA viruses.

This study visualizes the interior of a dsRNA virus using cryo-electron microscopy, revealing the organization of the genome of cytoplasmic polyhedrosis virus together with its transcriptional enzyme complex in both quiescent and transcribing states. Genome packing in a dsRNA virus Genome packaging in double-stranded RNA viruses is poorly understood. Using direct electron-counting cryoelectron microscopy and asymmetric reconstruction, Hong Zhou and colleagues visualize in situ structures of the genome of insect cytoplasmic polyhedrosis virus (CPV) in quiescent and transcribing states. The structures reveal that each CPV capsid contains ten segmented dsRNAs, organized with ten transcriptional enzyme complexes in a specific, non-symmetric manner, with each dsRNA segment attached directly to a transcriptional enzyme complex. Viruses in the Reoviridae , like the triple-shelled human rotavirus and the single-shelled insect cytoplasmic polyhedrosis virus (CPV), all package a genome of segmented double-stranded RNAs (dsRNAs) inside the viral capsid and carry out endogenous messenger RNA synthesis through a transcriptional enzyme complex (TEC) 1 . By direct electron-counting cryoelectron microscopy and asymmetric reconstruction, we have determined the organization of the dsRNA genome inside quiescent CPV (q-CPV) and the in situ atomic structures of TEC within CPV in both quiescent and transcribing (t-CPV) states. We show that the ten segmented dsRNAs in CPV are organized with ten TECs in a specific, non-symmetric manner, with each dsRNA segment attached directly to a TEC. The TEC consists of two extensively interacting subunits: an RNA-dependent RNA polymerase (RdRP) and an NTPase VP4. We find that the bracelet domain of RdRP undergoes marked conformational change when q-CPV is converted to t-CPV, leading to formation of the RNA template entry channel and access to the polymerase active site. An amino-terminal helix from each of two subunits of the capsid shell protein (CSP) interacts with VP4 and RdRP. These findings establish the link between sensing of environmental cues by the external proteins and activation of endogenous RNA transcription by the TEC inside the virus.

Cas13 endonuclease activity depends on the RNA local secondary structure with strong preference for single-stranded (SS) regions. Hence , it becomes indispensable to identify the SS regions for effective Cas13 mediated RNA knockdown. We herein present rational gRNA design by integrating experimental structure-seq data and predicted structural models. Utilizing structure-seq data for XIST transcript, we observed that gRNAs targeting the SS regions significantly induce transcript knockdown and cleavage than those targeting double-stranded (DS) regions. Further, we identified the “central seed region” in the gRNA that upon targeting the SS regions efficiently facilitates Cas13 mediated cleavage. In our following pursuits, we considered the scenario wherein experimental structure-seq data is not available, hence we used SS18-SSX2 fusion transcript indicated in synovial sarcomas and computationally predicted its structure. We observed that gRNAs targeting the SS regions predicted from the structure, efficiently induced necrosis compared to gRNAs that target the DS regions. In conclusion, for the effective RNA knockdown, the Cas13 mediated targeting strategy presented herein emphasizes the designing of gRNAs specifically targeting SS regions by utilizing structural information. Further, this strategy, in turn, can be anticipated to narrow the search space for gRNA design (by exclusively targeting SS regions) especially when lncRNAs are the targets.

Double-stranded RNA (dsRNA) viruses possess a segmented dsRNA genome and a number of RNA-dependent RNA polymerases (RdRps) enclosed in a capsid. Until now, the precise structures of genomes and RdRps within the capsids have been unknown. Here we report the structures of RdRps and associated RNAs within nontranscribing and transcribing cypoviruses (NCPV and TCPV, respectively), using a combination of cryo–electron microscopy (cryo-EM) and a symmetry-mismatch reconstruction method. The RdRps and associated RNAs appear to exhibit a pseudo-D3 symmetric organization in both NCPV and TCPV. However, the molecular interactions between RdRps and the genomic RNA were found to differ in these states. Our work provides insight into the mechanisms of the replication and transcription in dsRNA viruses and paves a way for structural determination of lower-symmetry complexes enclosed in higher-symmetry structures.

Human Dicer can process long double-stranded RNA and hairpin precursor RNA to yield short interfering RNAs or microRNAs, respectively. EM and single-particle analyses of Dicer–substrate complexes now provide insight into the structural basis of Dicer's substrate preference, implicating RNA structure and cofactors in determining substrate recognition and processing efficiency by Dicer. Dicer has a central role in RNA-interference pathways by cleaving double-stranded RNAs (dsRNAs) to produce small regulatory RNAs. Human Dicer can process long double-stranded and hairpin precursor RNAs to yield short interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. Previous studies have shown that pre-miRNAs are cleaved more rapidly than pre-siRNAs in vitro and are the predominant natural Dicer substrates. We have used EM and single-particle analysis of Dicer–RNA complexes to gain insight into the structural basis for human Dicer's substrate preference. Our studies show that Dicer traps pre-siRNAs in a nonproductive conformation, whereas interactions of Dicer with pre-miRNAs and dsRNA-binding proteins induce structural changes in the enzyme that enable productive substrate recognition in the central catalytic channel. These findings implicate RNA structure and cofactors in determining substrate recognition and processing efficiency by human Dicer.

Melanoma differentiation-associated protein 5 (MDA5) detects viral dsRNA in the cytoplasm. On binding of RNA, MDA5 forms polymers, which trigger assembly of the signaling adaptor mitochondrial antiviral-signaling protein (MAVS) into its active fibril form. The molecular mechanism of MDA5 signaling is not well understood, however. Here we show that MDA5 forms helical filaments on dsRNA and report the 3D structure of the filaments using electron microscopy (EM) and image reconstruction. MDA5 assembles into a polar, singlestart helix around the RNA. Fitting of an MDA5 homology model into the structure suggests a key role for the MDA5 C-terminal domain in cooperative filament assembly. Our study supports a signal transduction mechanism in which the helical array of MDA5 within filaments nucleates the assembly of MAVS fibrils. We conclude that MDA5 is a polymerization-dependent signaling platform that uses the amyloid-like self-propagating properties of MAVS to amplify signaling.

Viruses evolve so rapidly that sequence-based comparison is not suitable for detecting relatedness among distant viruses. Structure-based comparisons suggest that evolution led to a small number of viral classes or lineages that can be grouped by capsid protein (CP) folds. Here, we report that the CP structure of the fungal dsRNA Penicillium chrysogenum virus (PcV) shows the progenitor fold of the dsRNA virus lineage and suggests a relationship between lineages. Cryo-EM structure at near-atomic resolution showed that the 982-aa PcV CP is formed by a repeated α-helical core, indicative of gene duplication despite lack of sequence similarity between the two halves. Superimposition of secondary structure elements identified a single “hotspot” at which variation is introduced by insertion of peptide segments. Structural comparison of PcV and other distantly related dsRNA viruses detected preferential insertion sites at which the complexity of the conserved α-helical core, made up of ancestral structural motifs that have acted as a skeleton, might have increased, leading to evolution of the highly varied current structures. Analyses of structural motifs only apparent after systematic structural comparisons indicated that the hallmark fold preserved in the dsRNA virus lineage shares a long (spinal) α-helix tangential to the capsid surface with the head-tailed phage and herpesvirus viral lineage.