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Long non-coding RNAs (lncRNAs) constitute a significant fraction of the transcriptome, playing important roles in development and disease. However, our understanding of structure-function relationships for this emerging class of RNAs has been limited to secondary structures. Here, we report the 3-D atomistic structural study of epigenetic lncRNA, Braveheart (Bvht) , and its complex with CNBP (Cellular Nucleic acid Binding Protein). Using small angle X-ray scattering (SAXS), we elucidate the ensemble of Bvht RNA conformations in solution, revealing that Bvht lncRNA has a well-defined, albeit flexible 3-D structure that is remodeled upon CNBP binding. Our study suggests that CNBP binding requires multiple domains of Bvht and the RHT/AGIL RNA motif. We show that RHT/AGIL, previously shown to interact with CNBP, contains a highly flexible loop surrounded by more ordered helices. As one of the largest RNA-only 3-D studies, the work lays the foundation for future structural studies of lncRNA-protein complexes. Many RNA systems possess highly ordered 3-D structures that are essential to their function. Here the authors demonstrate that the long non-coding RNA Braveheart possesses a flexible but defined 3-D structure which is remodeled upon binding the protein CNBP.

RNA viruses have evolved numerous strategies to overcome host resistance and immunity, including the use of multifunctional proteases that not only cleave viral polyproteins during virus replication but also deubiquitinate cellular proteins to suppress ubiquitin (Ub)-mediated antiviral mechanisms. Here, we report an approach to attenuate the infection of Arabidopsis thaliana by Turnip Yellow Mosaic Virus (TYMV) by suppressing the polyprotein cleavage and deubiquitination activities of the TYMV protease (PRO). Performing selections using a library of phage-displayed Ub variants (UbVs) for binding to recombinant PRO yielded several UbVs that bound the viral protease with nanomolar affinities and blocked its function. The strongest binding UbV (UbV3) candidate had a EC 50 of 0.3 nM and inhibited both polyprotein cleavage and DUB activity of PRO in vitro . X-ray crystal structures of UbV3 alone and in complex with PRO reveal that the inhibitor exists as a dimer that binds two copies of PRO. Consistent with our biochemical and structural findings, transgenic expression of UbV3 in the cytosol of A . thaliana suppressed TYMV replication in planta , with the reduction in viral load being correlated to UbV3 expression level. Our results demonstrate the potential of using UbVs to protect plants from tymovirus infection, a family of viruses that contain numerous members of significant agricultural concern, as well as other plant viruses that express functionally related proteases with deubiquitinating activity.

The hepatitis B virus (HBV) chronically infects over 250 million people worldwide and is one of the leading causes of liver cancer and hepatocellular carcinoma. HBV persistence is due in part to the highly stable HBV minichromosome or HBV covalently closed circular DNA (cccDNA) that resides in the nucleus. As HBV replication requires the help of host transcription factors to replicate, focusing on host protein–HBV genome interactions may reveal insights into new drug targets against cccDNA. The structural details on such complexes, however, remain poorly defined. In this review, the current literature regarding host transcription factors’ interactions with HBV cccDNA is discussed.

Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.

Netrins, a family of laminin-related molecules, have been proposed to act as guidance cues either during nervous system development or the establishment of the vascular system. This was clearly demonstrated for netrin-1 via its interaction with the receptors DCC and UNC5s. However, mainly based on shared homologies with netrin-1, netrin-4 was also proposed to play a role in neuronal outgrowth and developmental/pathological angiogenesis via interactions with netrin-1 receptors. Here, we present the high-resolution structure of netrin-4, which shows unique features in comparison with netrin-1, and show that it does not bind directly to any of the known netrin-1 receptors. We show that netrin-4 disrupts laminin networks and basement membranes (BMs) through high-affinity binding to the laminin γ1 chain. We hypothesize that this laminin-related function is essential for the previously described effects on axon growth promotion and angiogenesis. Our study unveils netrin-4 as a non-enzymatic extracellular matrix protein actively disrupting pre-existing BMs. Netrins are secreted guidance factors that promote axon outgrowth and orientation during nervous system development. Here the authors present structural and biological evidence that netrin-4 is not a guidance cue per se, but rather functions to modulate laminin-laminin interactions.

Seaweeds represent a promising source of sustainable, alternative feeds for livestock. Despite their increasing popularity in agriculture, the dietary fate of seaweed polysaccharides, such as carrageenan, is unknown. Here, we apply functional microbiome analyses of ruminant gastrointestinal tract microbiomes to discover catabolic enzymes specific for carrageenan digestion from the red seaweed Mazzaella japonica . M. japonica preferentially increased Bacteroides abundance within the feces over the rumen, and bacterial isolates have the capacity to use carrageenans as a sole carbon source. We identify carrageenan-active polysaccharide utilization loci (CarPULs) and characterize recombinant GH16 subfamily 17 carrageenases, informing previously uncharacterized substrate specificities for the subfamily, and providing insights into pathway specialization of divergent CarPULs. Selective enrichment and metagenomic mining reveals that carrageenan catabolism is widespread among geographically and taxonomically distinct ruminants, suggesting it is a latent trait widely distributed in the Order Artiodactyla and carried within their microbiomes as part of the microbial “dark matter”. These pathways are structurally distinct from those found in marine bacteria, highlighting a complex and ancient evolutionary history of CarPULs in ruminant microbiomes. Although seaweed is a sustainable feed option for cattle, it’s not known if its cell wall polysaccharides are digested. Here authors show the seaweed Mazzaella japonica enriches for carrageenan-digesting bacteria in the lower gut of diverse ruminants.

Highly structured RNA derived from viral genomes is a key cellular indicator of viral infection. In response, cells produce the interferon inducible RNA-dependent protein kinase (PKR) that, when bound to viral dsRNA, phosphorylates eukaryotic initiation factor 2α and attenuates viral protein translation. Adenovirus can evade this line of defence through transcription of a non-coding RNA, VAI, an inhibitor of PKR. VAI consists of three base-paired regions that meet at a three-way junction; an apical stem responsible for the interaction with PKR, a central stem required for inhibition, and a terminal stem. Recent studies have highlighted the potential importance of the tertiary structure of the three-way junction to PKR inhibition by enabling interaction between regions of the central and terminal stems. To further investigate the role of the three-way junction, we characterized the binding affinity and inhibitory potential of central stem mutants designed to introduce subtle alterations. These results were then correlated with small-angle X-ray scattering solution studies and computational tertiary structural models. Our results demonstrate that while mutations to the central stem have no observable effect on binding affinity to PKR, mutations that appear to disrupt the structure of the three-way junction prevent inhibition of PKR. Therefore, we propose that instead of simply sequestering PKR, a specific structural conformation of the PKR-VAI complex may be required for inhibition.

Zika virus (ZIKV) infection causes significant human disease that, with no approved treatment or vaccine, constitutes a major public health concern. Its life cycle entirely relies on the cytoplasmic fate of the viral RNA genome (vRNA) through a fine-tuned equilibrium between vRNA translation, replication, and packaging into new virions, all within virus-induced replication organelles (vROs). In this study, with an RNA interference (RNAi) mini-screening and subsequent functional characterization, we have identified insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) as a new host dependency factor that regulates vRNA synthesis. In infected cells, IGF2BP2 associates with viral NS5 polymerase and redistributes to the perinuclear viral replication compartment. Combined fluorescence in situ hybridization-based confocal imaging, in vitro binding assays, and immunoprecipitation coupled to RT-qPCR showed that IGF2BP2 directly interacts with ZIKV vRNA 3’ nontranslated region. Using ZIKV sub-genomic replicons and a replication-independent vRO induction system, we demonstrated that IGF2BP2 knockdown impairs de novo vRO biogenesis and, consistently, vRNA synthesis. Finally, the analysis of immunopurified IGF2BP2 complex using quantitative mass spectrometry and RT-qPCR revealed that ZIKV infection alters the protein and RNA interactomes of IGF2BP2. Altogether, our data support that ZIKV hijacks and remodels the IGF2BP2 ribonucleoprotein complex to regulate vRO biogenesis and vRNA neosynthesis.

Hantaviruses, part of the Bunyaviridae family, are a genus of negative-sense, single-stranded RNA viruses that cause two major diseases: New-World Hantavirus Cardiopulmonary Syndrome and Old-World Hemorrhagic Fever with Renal Syndrome. Hantaviruses generally are found worldwide with each disease corresponding to their respective hemispheres. New-World Hantaviruses spread by specific rodent-host reservoirs and are categorized as emerging viruses that pose a threat to global health and security due to their high mortality rate and ease of transmission. Incidentally, reports of Hantavirus categorization as a bioweapon are often contradicted as both US National Institute of Allergy and Infectious Diseases and the Centers for Disease Control and Prevention refer to them as Category A and C bioagents respectively, each retaining qualitative levels of importance and severity. Concerns of Hantavirus being engineered into a novel bioagent has been thwarted by Hantaviruses being difficult to culture, isolate, and purify limiting its ability to be weaponized. However, the natural properties of Hantaviruses pose a threat that can be exploited by conventional and unconventional forces. This review seeks to clarify the categorization of Hantaviruses as a bioweapon, whilst defining the practicality of employing New-World Hantaviruses and their effect on armies, infrastructure, and civilian targets.

Viral infections are responsible for numerous deaths worldwide. Flaviviruses, which contain RNA as their genetic material, are one of the most pathogenic families of viruses. There is an increasing amount of evidence suggesting that their 5’ and 3’ non-coding terminal regions are critical for their survival. Information on their structural features is essential to gain detailed insights into their functions and interactions with host proteins. In this study, the 5’ and 3’ terminal regions of Murray Valley encephalitis virus and Powassan virus were examined using biophysical and computational modeling methods. First, we used size exclusion chromatography and analytical ultracentrifuge methods to investigate the purity of in-vitro transcribed RNAs. Next, we employed small-angle X-ray scattering techniques to study solution conformation and low-resolution structures of these RNAs, which suggest that the 3’ terminal regions are highly extended as compared to the 5’ terminal regions for both viruses. Using computational modeling tools, we reconstructed 3-dimensional structures of each RNA fragment and compared them with derived small-angle X-ray scattering low-resolution structures. This approach allowed us to reinforce that the 5’ terminal regions adopt more dynamic structures compared to the mainly double-stranded structures of the 3’ terminal regions.