RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA three-dimensional structure prediction. With agreement from structural biologists, RNA structures are predicted by modeling groups before publication of the experimental structures. We report a large-scale set of predictions by 18 groups for 23 RNA-Puzzles: 4 RNA elements, 2 Aptamers, 4 Viral elements, 5 Ribozymes and 8 Riboswitches. We describe automatic assessment protocols for comparisons between prediction and experiment. Our analyses reveal some critical steps to be overcome to achieve good accuracy in modeling RNA structures: identification of helix-forming pairs and of non-Watson–Crick modules, correct coaxial stacking between helices and avoidance of entanglements. Three of the top four modeling groups in this round also ranked among the top four in the CASP15 contest.

The extreme 5′-end of the enterovirus RNA genome contains a conserved cloverleaf-like domain that recruits 3CD and PCBP proteins required for initiating genome replication. Here, we report the crystal structure at 1.9 Å resolution of this domain from the CVB3 genome in complex with an antibody chaperone. The RNA folds into an antiparallel H-type four-way junction comprising four subdomains with co-axially stacked sA-sD and sB-sC helices. Long-range interactions between a conserved A40 in the sC-loop and Py-Py helix within the sD subdomain organize near-parallel orientations of the sA-sB and sC-sD helices. Our NMR studies confirm that these long-range interactions occur in solution and without the chaperone. The phylogenetic analyses indicate that our crystal structure represents a conserved architecture of enteroviral cloverleaf-like domains, including the A40 and Py-Py interactions. The protein binding studies further suggest that the H-shape architecture provides a ready-made platform to recruit 3CD and PCBP2 for viral replication.

The genomes of several plant viruses contain RNA structures at their 3′ ends called cap-independent translation enhancers (CITEs) that bind the host protein factors such as mRNA 5′ cap-binding protein eIF4E for promoting cap-independent genome translation. However, the structural basis of such 5′ cap-binding protein recognition by the uncapped RNA remains largely unknown. Here, we have determined the crystal structure of a 3′ CITE, panicum mosaic virus-like translation enhancer (PTE) from the saguaro cactus virus (SCV), using a Fab crystallization chaperone. The PTE RNA folds into a three-way junction architecture with a pseudoknot between the purine-rich R domain and pyrimidine-rich Y domain, which organizes the overall structure to protrude out a specific guanine nucleotide, G18, from the R domain that comprises a major interaction site for the eIF4E binding. The superimposable crystal structures of the wild-type, G18A, G18C, and G18U mutants suggest that the PTE scaffold is preorganized with the flipped-out G18 ready to dock into the eIF4E 5′ cap-binding pocket. The binding studies with wheat and human eIF4Es using gel electrophoresis and isothermal titration calorimetry, and molecular docking computation for the PTE–eIF4E complex demonstrated that the PTE structure essentially mimics the mRNA 5′ cap for eIF4E binding. Such 5′ cap mimicry by the uncapped and structured viral RNA highlights how viruses can exploit RNA structures to mimic the host protein-binding partners and bypass the canonical mechanisms for their genome translation, providing opportunities for a better understanding of virus-host interactions and non-canonical translation mechanisms found in many pathogenic RNA viruses.

With increasing climate variability, a sustainable crop production approach remains an indispensable concern across the globe. In this study, P retention/availability of MgCl₂.6H₂O/KOH modified Douglas fir biochar was assessed. The MgCl₂·6H₂O/KOH treated Douglas fir biochar was prepared by sequentially treating Douglas fir biochar with magnesium chloride and potassium hydroxide solutions. The biochar’s surface area, pore volume, morphology, and elemental compositions were determined using BET, SEM, SEM/EDS, and powder X-ray analyzes. Both surface area and pore volume were reduced by more than 97% following modification. Similarly, the morphology and elemental compositions changed after modification. The maximum P adsorbed corresponding to Langmuir–Freundlich model was 41.18 mg g⁻¹. P sorption on biochar soil mixture was pH dependent. More studies are required to establish the field applicability of P-laden MgCl₂ ·6H₂O/KOH-modified Douglas fir biochar as a soil additive.

RNA domains within viral IRESs are crucial for initiating cap-independent translation of the genome in many positive-sense RNA viruses. However, the structures and mechanisms of these IRES domains remain unclear. Here, we present the 3 Å resolution crystal structure of the coxsackievirus B3 (CVB3) IRES domain V (dV) as a model for type I IRESs. The crystal structure revealed an elongated architecture of dV, with two sets of coaxially stacked stems forming an H-type four-way junction (4WJ) organized by an A-rich motif. Despite sequence dissimilarities, this dV from a type I IRES exhibits remarkable structural similarity to the analogous tertiary structures of the encephalomyocarditis virus (EMCV) JK domain and the hepatitis A virus (HAV) dV, which are typical domains in the type II and III IRESs, respectively. While SAXS studies indicate a similar RNA fold of dV in solution, structure-guided binding, computational modeling, and X-ray footprinting studies with and without the HEAT1 domain of eIF4G, compared to the analogous type II (EMCV JK) and III (HAV dV) domains, suggest that various IRESs maintain a common mechanism of eIF4G binding interactions during viral genome translation. Despite sequence variability, this structural conservation across IRES types may offer unique opportunities to develop universal antivirals targeting these structures.

Although antibody derivatives, such as Fabs and scFvs, have revolutionized the cellular imaging, quantification and tracking of proteins, analogous tools and strategies are unavailable for cellular RNA visualization. Here, we developed four synthetic anti-RNA scFv (sarabody) probes and their green fluorescent protein (GFP) fusions and demonstrated their potential to visualize RNA in live mammalian cells. We expressed these sarabodies and sarabody–GFP modules, purified them as soluble proteins, characterized their binding interactions with their corresponding epitopes and finally employed two of the four modules, sara1-GFP and sara1c-GFP, to visualize a target messenger RNA in live U2OS cells. Our current RNA imaging strategy is analogous to the existing MCP-MS2 system for RNA visualization, but additionally, our approach provides robust flexibility for developing target RNA-specific imaging modules, as epitope-specific probes can be selected from a library generated by diversifying the sarabody complementarity determining regions. While we continue to optimize these probes, develop new probes for various target RNAs and incorporate other fluorescence proteins like mCherry and HaloTag, our groundwork results demonstrated that these first-of-a-kind immunofluorescent probes will have tremendous potential for tracking mature RNAs and may aid in visualizing and quantifying many cellular processes as well as examining the spatiotemporal dynamics of various RNAs.