The In Vivo and In Vitro Architecture of the Hepatitis C Virus RNA Genome Uncovers Functional RNA Secondary and Tertiary Structures.

Hepatitis C virus (HCV) is a positive-strand RNA virus that remains one of the main contributors to chronic liver disease worldwide. Studies over the last 30 years have demonstrated that HCV contains a highly structured RNA genome and many of these structures play essential roles in the HCV life cycle. Despite the importance of riboregulation in this virus, most of the HCV RNA genome remains functionally unstudied. Here, we report a complete secondary structure map of the HCV RNA genome in vivo, which was studied in parallel with the secondary structure of the same RNA obtained in vitro. Our results show that HCV is folded extensively in the cellular context. By performing comprehensive structural analyses on both in vivo data and in vitro data, we identify compact and conserved secondary and tertiary structures throughout the genome. Genetic and evolutionary functional analyses demonstrate that many of these elements play important roles in the virus life cycle. In addition to providing a comprehensive map of RNA structures and riboregulatory elements in HCV, this work provides a resource for future studies aimed at identifying therapeutic targets and conducting further mechanistic studies on this important human pathogen. IMPORTANCE HCV has one of the most highly structured RNA genomes studied to date, and it is a valuable model system for studying the role of RNA structure in protein-coding genes. While previous studies have identified individual cases of regulatory RNA structures within the HCV genome, the full-length structure of the HCV genome has not been determined in vivo. Here, we present the complete secondary structure map of HCV determined both in cells and from corresponding transcripts generated in vitro. In addition to providing a comprehensive atlas of functional secondary structural elements throughout the genomic RNA, we identified a novel set of tertiary interactions and demonstrated their functional importance. In terms of broader implications, the pipeline developed in this study can be applied to other long RNAs, such as long noncoding RNAs. In addition, the RNA structural motifs characterized in this study broaden the repertoire of known riboregulatory elements.

Dbp5 associates with RNA-bound Mex67 and Nab2 and its localization at the nuclear pore complex is sufficient for mRNP export and cell viability.

In Saccharomyces cerevisiae, the mRNA export receptor Mex67 is recruited to mature nuclear transcripts to mediate mRNA export through the nuclear pore complex (NPC) to the cytoplasm. Mex67 binds transcripts through adaptor proteins such as the poly(A) binding protein Nab2. When a transcript reaches the cytoplasmic face of the NPC, the DEAD-box protein Dbp5 acts to induce a local structural change to release Nab2 and Mex67 in an essential process termed mRNP remodeling. It is unknown how certain proteins (Nab2, Mex67) are released during Dbp5-mediated mRNP remodeling, whereas others remain associated. Here, we demonstrate that Dbp5 associates in close proximity with Mex67 and Nab2 in a cellular complex. Further, fusion of Dbp5 to Nup159 anchors Dbp5 at the cytoplasmic face of the NPC and is sufficient for cell viability. Thus, we speculate that the essential role of Dbp5 in remodeling exporting mRNPs requires its localization to the NPC and is separable from other subcellular functions of Dbp5. This work supports a model where the diverse nuclear, cytoplasmic and NPC functions of Dbp5 in the mRNA lifecycle are not interdependent and that Dbp5 is locally recruited through complex protein-protein interactions to select regions of transcripts for specific removal of transport proteins at the NPC.

Sequencing and Structure Probing of Long RNAs Using MarathonRT: A Next-Generation Reverse Transcriptase.

Reverse transcriptase (RT) enzymes are indispensable tools for interrogating diverse aspects of RNA metabolism and transcriptome composition. Due to the growing interest in sequence and structural complexity of long RNA molecules, processive RT enzymes are now required for preserving linkage and information content in mixed populations of transcripts, and the low-processivity RT enzymes that are commercially available cannot meet this need. MarathonRT is encoded within a eubacterial group II intron, and it has been shown to efficiently copy highly structured long RNA molecules in a single pass. In this work, we systematically characterize MarathonRT as a tool enzyme and optimize its performance in a variety of applications that include single-cycle reverse transcription of long RNAs, dimethyl sulfate mutational profiling (DMS-MaP), selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), using ultra-long amplicons and the detection of natural RNA base modifications. By diversifying MarathonRT reaction protocols, we provide an upgraded suite of tools for cutting-edge RNA research and clinical application.

Sensitive detection of structural features and rearrangements in long, structured RNA molecules.

Technical innovations in structural probing have drastically advanced the field of RNA structure analysis. These advances have led to parallel approaches developed in separate labs for analyzing RNA structure and dynamics. With the wealth of methodologies available, it can be difficult to determine which is best suited for a given application. Here, using a long, highly structured viral RNA as an example (the positive strand genome of Hepatitis C Virus), we present a semi-comprehensive analysis and describe the major approaches for analyzing the architecture of RNA that is modified with structure-sensitive probes. Additionally, we present an updated method for generating in vitro transcribed and folded RNA that maintains native secondary structures in long RNA molecules. We anticipate that the methods described here will streamline the use of current approaches and help investigators who are unfamiliar with structure probing, obviating the need for time-consuming and expensive optimization.

Reply to "Comment on: Comprehensive Nutritional and Dietary Intervention for Autism Spectrum Disorder-A Randomized, Controlled 12-Month Trial, Nutrients 2018, 10, 369".

We thank Vorland et al [...].

Small molecules that target group II introns are potent antifungal agents.

Specific RNA structures control numerous metabolic processes that impact human health, and yet efforts to target RNA structures de novo have been limited. In eukaryotes, the self-splicing group II intron is a mitochondrial RNA tertiary structure that is absent in vertebrates but essential for respiration in plants, fungi and yeast. Here we show that this RNA can be targeted through a process of high-throughput in vitro screening, SAR and lead optimization, resulting in high-affinity compounds that specifically inhibit group IIB intron splicing in vitro and in vivo and lack toxicity in human cells. The compounds are potent growth inhibitors of the pathogen Candida parapsilosis, displaying antifungal activity comparable to that of amphotericin B. These studies demonstrate that RNA tertiary structures can be successfully targeted de novo, resulting in pharmacologically valuable compounds.

microRNA-122 amplifies hepatitis C virus translation by shaping the structure of the internal ribosomal entry site.

The liver-specific microRNA-122 (miR-122) recognizes two conserved sites at the 5' end of the hepatitis C virus (HCV) genome and contributes to stability, translation, and replication of the viral RNA. We show that stimulation of the HCV internal ribosome entry site (IRES) by miR-122 is essential for efficient viral replication. The mechanism relies on a dual function of the 5' terminal sequence in the complementary positive (translation) and negative strand (replication), requiring different secondary structures. Predictions and experimental evidence argue for several alternative folds involving the miR-binding region (MBR) adjacent to the IRES and interfering with its function. Mutations in the MBR, designed to suppress these dysfunctional structures indeed stimulate translation independently of miR-122. Conversely, MBR mutants favoring alternative folds show impaired IRES activity. Our results therefore suggest that miR-122 binding assists the folding of a functional IRES in an RNA chaperone-like manner by suppressing energetically favorable alternative secondary structures.

Comprehensive Nutritional and Dietary Intervention for Autism Spectrum Disorder-A Randomized, Controlled 12-Month Trial.

This study involved a randomized, controlled, single-blind 12-month treatment study of a comprehensive nutritional and dietary intervention. Participants were 67 children and adults with autism spectrum disorder (ASD) ages 3-58 years from Arizona and 50 non-sibling neurotypical controls of similar age and gender. Treatment began with a special vitamin/mineral supplement, and additional treatments were added sequentially, including essential fatty acids, Epsom salt baths, carnitine, digestive enzymes, and a healthy gluten-free, casein-free, soy-free (HGCSF) diet. There was a significant improvement in nonverbal intellectual ability in the treatment group compared to the non-treatment group (+6.7 ± 11 IQ points vs. -0.6 ± 11 IQ points, p = 0.009) based on a blinded clinical assessment. Based on semi-blinded assessment, the treatment group, compared to the non-treatment group, had significantly greater improvement in autism symptoms and developmental age. The treatment group had significantly greater increases in EPA, DHA, carnitine, and vitamins A, B2, B5, B6, B12, folic acid, and Coenzyme Q10. The positive results of this study suggest that a comprehensive nutritional and dietary intervention is effective at improving nutritional status, non-verbal IQ, autism symptoms, and other symptoms in most individuals with ASD. Parents reported that the vitamin/mineral supplements, essential fatty acids, and HGCSF diet were the most beneficial.

NS3 from Hepatitis C Virus Strain JFH-1 Is an Unusually Robust Helicase That Is Primed To Bind and Unwind Viral RNA.

Hepatitis C viruses (HCV) encode a helicase enzyme that is essential for viral replication and assembly (nonstructural protein 3 [NS3]). This helicase has become the focus of extensive basic research on the general helicase mechanism, and it is also of interest as a novel drug target. Despite the importance of this protein, mechanistic work on NS3 has been conducted almost exclusively on variants from HCV genotype 1. Our understanding of NS3 from the highly active HCV strains that are used to study HCV genetics and mechanism in cell culture (such as JFH-1) is lacking. We therefore set out to determine whether NS3 from the replicatively efficient genotype 2a strain JFH-1 displays novel functional or structural properties. Using biochemical assays for RNA binding and duplex unwinding, we show that JFH-1 NS3 binds RNA much more rapidly than the previously studied NS3 variants from genotype 1b. Unlike NS3 variants from other genotypes, JFH-1 NS3 binds RNA with high affinity in a functionally active form that is capable of immediately unwinding RNA duplexes without undergoing rate-limiting conformational changes that precede activation. Unlike other superfamily 2 (SF2) helicases, JFH-1 NS3 does not require long 3' overhangs, and it unwinds duplexes that are flanked by only a few nucleotides, as in the folded HCV genome. To understand the physical basis for this, we solved the crystal structure of JFH-1 NS3, revealing a novel conformation that contains an open, positively charged RNA binding cleft that is primed for productive interaction with RNA targets, potentially explaining robust replication by HCV JFH-1.IMPORTANCE Genotypes of HCV are as divergent as different types of flavivirus, and yet mechanistic features of HCV variants are presumed to be held in common. One of the most well-studied components of the HCV replication complex is a helicase known as nonstructural protein 3 (NS3). We set out to determine whether this important mechanical component possesses biochemical and structural properties that differ between common strains such as those of genotype 1b and a strain of HCV that replicates with exceptional efficiency (JFH-1, classified as genotype 2a). Indeed, unlike the inefficient genotype 1b NS3, which has been well studied, JFH-1 NS3 is a superhelicase with strong RNA affinity and high unwinding efficiency on a broad range of targets. Crystallographic analysis reveals architectural features that promote enhanced biochemical activity of JFH-1 NS3. These findings show that even within a single family of viruses, drift in sequence can result in the acquisition of radically new functional properties that enhance viral fitness.

Nup42 and IP6 coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells.

The mRNA lifecycle is driven through spatiotemporal changes in the protein composition of mRNA particles (mRNPs) that are triggered by RNA-dependent DEAD-box protein (Dbp) ATPases. As mRNPs exit the nuclear pore complex (NPC) in Saccharomyces cerevisiae, this remodeling occurs through activation of Dbp5 by inositol hexakisphosphate (IP6 )-bound Gle1. At the NPC, Gle1 also binds Nup42, but Nup42's molecular function is unclear. Here we employ the power of structure-function analysis in S. cerevisiae and human (h) cells, and find that the high-affinity Nup42-Gle1 interaction is integral to Dbp5 (hDDX19B) activation and efficient mRNA export. The Nup42 carboxy-terminal domain (CTD) binds Gle1/hGle1B at an interface distinct from the Gle1-Dbp5/hDDX19B interaction site. A nup42-CTD/gle1-CTD/Dbp5 trimeric complex forms in the presence of IP6 . Deletion of NUP42 abrogates Gle1-Dbp5 interaction, and disruption of the Nup42 or IP6 binding interfaces on Gle1/hGle1B leads to defective mRNA export in S. cerevisiae and human cells. In vitro, Nup42-CTD and IP6 stimulate Gle1/hGle1B activation of Dbp5 and DDX19B recombinant proteins in similar, nonadditive manners, demonstrating complete functional conservation between humans and S. cerevisiae. Together, a highly conserved mechanism governs spatial coordination of mRNP remodeling during export. This has implications for understanding human disease mutations that perturb the Nup42-hGle1B interaction.

Functional RNA structures throughout the Hepatitis C Virus genome.

The single-stranded Hepatitis C Virus (HCV) genome adopts a set of elaborate RNA structures that are involved in every stage of the viral lifecycle. Recent advances in chemical probing, sequencing, and structural biology have facilitated analysis of RNA folding on a genome-wide scale, revealing novel structures and networks of interactions. These studies have underscored the active role played by RNA in every function of HCV and they open the door to new types of RNA-targeted therapeutics.

A Novel Saccharomyces cerevisiae FG Nucleoporin Mutant Collection for Use in Nuclear Pore Complex Functional Experiments.

FG nucleoporins (Nups) are the class of proteins that both generate the permeability barrier and mediate selective transport through the nuclear pore complex (NPC). The FG Nup family has 11 members in Saccharomyces cerevisiae, and the study of mutants lacking different FG domains has been instrumental in testing transport models. To continue analyzing the distinct functional roles of FG Nups in vivo, additional robust genetic tools are required. Here, we describe a novel collection of S. cerevisiae mutant strains in which the FG domains of different groups of Nups are absent (Δ) in the greatest number documented to date. Using this plasmid-based ΔFG strategy, we find that a GLFG domain-only pore is sufficient for viability. The resulting extensive plasmid and strain resources are available to the scientific community for future in-depth in vivo studies of NPC transport.

Nucleoporin FG domains facilitate mRNP remodeling at the cytoplasmic face of the nuclear pore complex.

Directional export of messenger RNA (mRNA) protein particles (mRNPs) through nuclear pore complexes (NPCs) requires multiple factors. In Saccharomyces cerevisiae, the NPC proteins Nup159 and Nup42 are asymmetrically localized to the cytoplasmic face and have distinct functional domains: a phenylalanine-glycine (FG) repeat domain that docks mRNP transport receptors and domains that bind the DEAD-box ATPase Dbp5 and its activating cofactor Gle1, respectively. We speculated that the Nup42 and Nup159 FG domains play a role in positioning mRNPs for the terminal mRNP-remodeling steps carried out by Dbp5. Here we find that deletion (Δ) of both the Nup42 and Nup159 FG domains results in a cold-sensitive poly(A)+ mRNA export defect. The nup42ΔFG nup159ΔFG mutant also has synthetic lethal genetic interactions with dbp5 and gle1 mutants. RNA cross-linking experiments further indicate that the nup42ΔFG nup159ΔFG mutant has a reduced capacity for mRNP remodeling during export. To further analyze the role of these FG domains, we replaced the Nup159 or Nup42 FG domains with FG domains from other Nups. These FG "swaps" demonstrate that only certain FG domains are functional at the NPC cytoplasmic face. Strikingly, fusing the Nup42 FG domain to the carboxy-terminus of Gle1 bypasses the need for the endogenous Nup42 FG domain, highlighting the importance of proximal positioning for these factors. We conclude that the Nup42 and Nup159 FG domains target the mRNP to Gle1 and Dbp5 for mRNP remodeling at the NPC. Moreover, these results provide key evidence that character and context play a direct role in FG domain function and mRNA export.

Prognostic value of ZAP-70 expression in chronic lymphocytic leukemia as assessed by quantitative polymerase chain reaction and flow cytometry.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a disorder in which the tempo of disease progression is highly variable, and prognostic markers that can be utilized at diagnosis are regarded as clinically important. Currently, there are several prognostic factors, such as immunoglobulin heavy chain (IgVH) mutational status, and ZAP-70 protein expression in neoplastic B-cells, that have demonstrated significant discriminative power in the prognostication of CLL. They are, however, largely unavailable in the routine diagnostic laboratory setting. METHODS: In this study, we characterized the IgVH status and ZAP-70 expression by molecular techniques in a cohort of 108 patients with CLL, and correlated these results with three different methods of ZAP-70 expression by flow cytometry. We then assessed the results of these methods in terms of prognostic power as characterized by time to first treatment (TTFT). RESULTS: By comparing three different flow cytometry methods using receiver­operator curve (ROC) analysis, we identified that by utilizing a corrected mean fluorescence intensity (CorrMFI) algorithm for assessing ZAP-70 expression, there was good correlation with both IgVH mutational status, and ZAP-70 expression as assessed by qPCR. We were also able to show that ZAP-70 expression, as assessed by both qPCR and the CorrMFI method, was prognostic of TTFT. CONCLUSIONS: While confirmation in a larger patient cohort, with longer follow-up is required, we believe that the CorrMFI represents the most promising method currently available in a routine diagnostic setting for the assessment of ZAP-70 expression in CLL patients.

Safety and efficacy of deferasirox in the management of transfusion-dependent patients with myelodysplastic syndrome and aplastic anaemia: a perspective review.

Deferasirox is an orally administered, once-daily iron chelator with a generally good safety and efficacy profile. Reported adverse events in the older myelodysplastic population are somewhat different to the more intensively investigated and younger thalassaemic population. Renal impairment is the most concerning adverse event, but this is reversible if identified and the drug is withdrawn early. Gastrointestinal effects, particularly diarrhoea, can be troublesome for older patients, but can be minimized with tailored therapy. Negative iron balance can be achieved in most patients with a median dose of 20 mg/kg/day, and doses up to 40 mg/kg are possible in patients with severe iron overload, who are at risk of cardiac decompensation.

Uncovering nuclear pore complexity with innovation.

Advances in imaging and reductionist approaches have provided a high-resolution understanding of nuclear pore complex structure and transport, revealing unexpected mechanistic complexities based on nucleoporin functions and specialized import and export pathways.

Review article: Coagulation cascade and therapeutics update: relevance to nephrology. Part 1: Overview of coagulation, thrombophilias and history of anticoagulants.

Coagulation involves the regulated sequence of proteolytic activation of a series of zymogens to achieve appropriate and timely haemostasis in an injured vessel, in an environment that overwhelmingly favours an anticoagulant state. In the non-pathological state, the inciting event involves exposure of circulating factor VII/VIIa to extravascularly expressed tissue factor, which brings into motion the series of steps which results in amplification of the initial stimulus, culminating in the conversion of fibrinogen to fibrin and clot formation. The precisely synchronized cascade of events is counter-balanced by a system of anticoagulant mechanisms, which serve to ensure that the haemostatic effect is regulated and does not extend inappropriately. Conversely, in pathological states, these events can escape normal control mechanisms, due to either inherited or acquired defects, which lead to thrombosis. Current anticoagulant therapy, although based on medications that have been in existence for upwards of 80 years, is moving towards targeted therapy for specific coagulation factors and events in the coagulation cascade, based on the current knowledge of the main triggers and key events within the series of reactions that culminates in haemostasis. It remains to be seen whether these newer medications will become first-line therapies for thrombosis in the coming decade. This review aims to elucidate the main events within the coagulation cascade as it is currently understood to operate in vivo, with a brief discussion focusing on hypercoagulable states, and also a short review of the history of anticoagulants as they relate to this model.

Asymmetric membrane fiber-based carbon dioxide removal devices for ion chromatography.

The application of hollow and filament-filled single asymmetric membrane fibers, consisting of a thin silicone layer on a tubular microporous support, for removing CO(2) in suppressed ion chromatography effluents is described. With appropriate choice of the removal device and operating conditions, the CO(2) can be essentially quantitatively (99+%) removed. For carbonate-based eluents, the use of such devices greatly reduces or eliminates the water dip, permitting better quantitation of poorly retained anions that elute close to the dip, allows practical gradient chromatography, and improves noise levels and attainable detection limits. In hydroxide eluent chromatography, the device largely removes the response from CO(2) present in the samples; this greatly aids atmospheric trace gas analysis by IC. Device dimensions are such that the dispersion introduced by the device is small.