Structure of an RNA switch that enforces stringent retroviral genomic RNA dimerization

Retroviruses selectively package two copies of their RNA genomes in the context of a large excess of nongenomic RNA. Specific packaging of genomic RNA is achieved, in part, by recognizing RNAs that form a poorly understood dimeric structure at their 5′ ends. We identify, quantify the stability of, and use extensive experimental constraints to calculate a 3D model for a tertiary structure domain that mediates specific interactions between RNA genomes in a gamma retrovirus. In an initial interaction, two stem–loop structures from one RNA form highly stringent cross-strand loop–loop base pairs with the same structures on a second genomic RNA. Upon subsequent folding to the final dimer state, these intergenomic RNA interactions convert to a high affinity and compact tertiary structure, stabilized by interdigitated interactions between U-shaped RNA units. This retroviral conformational switch model illustrates how two-step formation of an RNA tertiary structure yields a stringent molecular recognition event at early assembly steps that can be converted to the stable RNA architecture likely packaged into nascent virions

Examining the ribonuclease H primer grip of HIV-1 reverse transcriptase by charge neutralization of RNA/DNA hybrids

The crystal structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) bound to an RNA/DNA hybrid reveals an extensive network of contacts with the phosphate backbone of the DNA strand ∼4–9 bp downstream from the ribonuclease H (RNase H) catalytic center. Collectively designated as ‘the RNase H primer grip’, this motif contains a phosphate binding pocket analogous to the human and Bacillus halodurans RNases H. The notion that the RNase H primer grip mediates the trajectory of RNA/DNA hybrids accessing the RNase H active site suggests that locally neutralizing the phosphate backbone may be exploited to manipulate nucleic acid flexibility. To examine this, we introduced single and tandem methylphosphonate substitutions through the region of the DNA primer contacted by the RNase H primer grip and into the RNase H catalytic center. The ability of mutant hybrids to support RNase H and DNA polymerase activity was thereafter examined. In addition, site-specific chemical footprinting was used to evaluate movement of the DNA polymerase and RNase H domains. We show here that minor alteration to the RNase H primer can have a dramatic effect on enzyme positioning, and discuss these findings in light of recent crystallography of human RNase H containing an RNA/DNA hybrid

Murine leukemia virus RNA dimerization is coupled to transcription and splicing processes

Most of the cell biological aspects of retroviral genome dimerization remain unknown. Murine leukemia virus (MLV) constitutes a useful model to study when and where dimerization occurs within the cell. For instance, MLV produces a subgenomic RNA (called SD') that is co-packaged with the genomic RNA predominantly as FLSD' heterodimers. This SD' RNA is generated by splicing of the genomic RNA and also by direct transcription of a splice-associated retroelement of MLV (SDARE). We took advantage of these two SD' origins to study the effects of transcription and splicing events on RNA dimerization. Using genetic approaches coupled to capture of RNA heterodimer in virions, we determined heterodimerization frequencies in different cellular contexts. Several cell lines were stably established in which SD' RNA was produced by either splicing or transcription from SDARE. Moreover, SDARE was integrated into the host chromosome either concomitantly or sequentially with the genomic provirus. Our results showed that transcribed genomic and SD' RNAs preferentially formed heterodimers when their respective proviruses were integrated together. In contrast, heterodimerization was strongly affected when the two proviruses were integrated independently. Finally, dimerization was enhanced when the transcription sites were expected to be physically close. For the first time, we report that splicing and RNA dimerization appear to be coupled. Indeed, when the RNAs underwent splicing, the FLSD' dimerization reached a frequency similar to co-transcriptional heterodimerization. Altogether, our results indicate that randomness of heterodimerization increases when RNAs are co-expressed during either transcription or splicing. Our results strongly support the notion that dimerization occurs in the nucleus, at or near the transcription and splicing sites, at areas of high viral RNA concentration

Exploring RNA Structural Codes with SHAPE Chemistry

RNA is the central conduit for gene expression. This role depends on an ability to encode information at two levels: in its linear sequence and in the complex structures RNA can form by folding back on itself. Understanding the global structure-function interrelationships mediated by RNA remains a great challenge in molecular and structural biology. In this Account, we discuss evolving work in our laboratory focused on creating facile, generic, quantitative, accurate, and highly informative approaches for understanding RNA structure in biologically important environments. The core innovation derives from our discovery that the nucleophilic reactivity of the ribose 2'-hydroxyl in RNA is gated by local nucleotide flexibility. The 2'-hydroxyl is reactive at conformationally flexible positions but is unreactive at nucleotides constrained by base pairing. Sites of modification in RNA can be detected efficiently either using primer extension or by protection from exoribonucleolytic degradation. This technology is now called SHAPE, for selective 2'-hydroxyl acylation analyzed by primer extension (or protection from exoribonuclease). SHAPE reactivities are largely independent of nucleotide identity but correlate closely with model-free measurements of molecular order. The simple SHAPE reaction is thus a robust, nucleotide-resolution, biophysical measurement of RNA structure. SHAPE can be used to provide an experimental correction to RNA folding algorithms and, in favorable cases, yield kilobase-scale secondary structure predictions with high accuracies. SHAPE chemistry is based on very simple reactive carbonyl centers that can be varied to yield slow- and fast-reacting reagents. Differential SHAPE reactivities can be used to detect specific RNA positions with slow local nucleotide dynamics. These positions, which are often in the C2'-endo conformation, have the potential to function as molecular timers that regulate RNA folding and function. In addition, fast-reacting SHAPE reagents can be used to visualize RNA structural biogenesis and RNA-protein assembly reactions in one second snapshots in very straightforward experiments. The application of SHAPE to challenging problems in biology has revealed surprises in well-studied systems. New regions have been identified that are likely to have critical functional roles on the basis of their high levels of RNA structure. For example, SHAPE analysis of large RNAs, such as authentic viral RNA genomes, suggests that RNA structure organizes regulatory motifs and regulates splicing, protein folding, genome recombination, and ribonucleoprotein assembly. SHAPE has also revealed limitations to the hierarchical model for RNA folding. Continued development and application of SHAPE technologies will advance our understanding of the many ways in which the genetic code is expressed through the underlying structure of RNA

Stratification of asthma phenotypes by airway proteomic signatures

© 2019 Background: Stratification by eosinophil and neutrophil counts increases our understanding of asthma and helps target therapy, but there is room for improvement in our accuracy in prediction of treatment responses and a need for better understanding of the underlying mechanisms. Objective: We sought to identify molecular subphenotypes of asthma defined by proteomic signatures for improved stratification. Methods: Unbiased label-free quantitative mass spectrometry and topological data analysis were used to analyze the proteomes of sputum supernatants from 246 participants (206 asthmatic patients) as a novel means of asthma stratification. Microarray analysis of sputum cells provided transcriptomics data additionally to inform on underlying mechanisms. Results: Analysis of the sputum proteome resulted in 10 clusters (ie, proteotypes) based on similarity in proteomic features, representing discrete molecular subphenotypes of asthma. Overlaying granulocyte counts onto the 10 clusters as metadata further defined 3 of these as highly eosinophilic, 3 as highly neutrophilic, and 2 as highly atopic with relatively low granulocytic inflammation. For each of these 3 phenotypes, logistic regression analysis identified candidate protein biomarkers, and matched transcriptomic data pointed to differentially activated underlying mechanisms. Conclusion: This study provides further stratification of asthma currently classified based on quantification of granulocytic inflammation and provided additional insight into their underlying mechanisms, which could become targets for novel therapies

International Consensus Statement on Allergy and Rhinology: Allergic Rhinitis

Background: Critical examination of the quality and validity of available allergic rhinitis (AR) literature is necessary to improve understanding and to appropriately translate this knowledge to clinical care of the AR patient. To evaluate the existing AR literature, international multidisciplinary experts with an interest in AR have produced the International Consensus statement on Allergy and Rhinology: Allergic Rhinitis (ICAR:AR).Methods: Using previously described methodology, specific topics were developed relating to AR. Each topic was assigned a literature review, evidence-based review (EBR), or evidence-based review with recommendations (EBRR) format as dictated by available evidence and purpose within the ICAR:AR document. Following iterative reviews of each topic, the ICAR:AR document was synthesized and reviewed by all authors for consensus.Results: The ICAR:AR document addresses over 100 individual topics related to AR, including diagnosis, pathophysiology, epidemiology, disease burden, risk factors for the development of AR, allergy testing modalities, treatment, and other conditions/comorbidities associated with AR.Conclusion: This critical review of the AR literature has identified several strengths; providers can be confident that treatment decisions are supported by rigorous studies. However, there are also substantial gaps in the AR literature. These knowledge gaps should be viewed as opportunities for improvement, as often the things that we teach and the medicine that we practice are not based on the best quality evidence. This document aims to highlight the strengths and weaknesses of the AR literature to identify areas for future AR research and improved understanding. </p

Structure of an RNA switch that enforces stringent retroviral genomic RNA dimerization

Retroviruses selectively package two copies of their RNA genomes in the context of a large excess of nongenomic RNA. Specific packaging of genomic RNA is achieved, in part, by recognizing RNAs that form a poorly understood dimeric structure at their 5′ ends. We identify, quantify the stability of, and use extensive experimental constraints to calculate a 3D model for a tertiary structure domain that mediates specific interactions between RNA genomes in a gamma retrovirus. In an initial interaction, two stem–loop structures from one RNA form highly stringent cross-strand loop–loop base pairs with the same structures on a second genomic RNA. Upon subsequent folding to the final dimer state, these intergenomic RNA interactions convert to a high affinity and compact tertiary structure, stabilized by interdigitated interactions between U-shaped RNA units. This retroviral conformational switch model illustrates how two-step formation of an RNA tertiary structure yields a stringent molecular recognition event at early assembly steps that can be converted to the stable RNA architecture likely packaged into nascent virions

CRTH2 antagonist, BI 671800 (BI), reduces nasal symptoms and inhibits nasal cytokines and eosinophils in SAR patients exposed to grass pollen in an environmental challenge chamber (ECC)

RATIONALE: Allergic rhinitis is a Th2 driven inflammatory disorder in which prostaglandin D2 (PGD2) is a key inflammatory component. PGD2 is released primarily by mast cells with TH2 cells, dendritic cells, and macrophages being secondary sources. PGD2 binds to its receptor (CRTH2) on basophils, mast cells, eosinophils, and TH2 cells releasing chemokine/inflammatory mediators. Targeting the CRTH2 receptor is a promising new therapy to reduce the allergic inflammation. METHODS: 146 grass sensitive SAR patients received one of 3 BI treatment doses (50, 200, 400 mg BID) or fluticasone propionate (FP) nasal spray 200 mcg or montelukast (MT) 10 mg daily for 2 weeks in a two way partial cross-over design. Each patient received placebo for their other 2 week treatment period. Subjects were exposed to Dactylis glomerata (4,000 grains/m3 over 6 h) in an ECC. Nasal symptoms (TNSS) were evaluated as the primary endpoint. Other endpoints included PGD2 induced eosinophil shape change (ESC), cytokines and eosinophils in nasal secretion and lavage. RESULTS: TNSS was significantly reduced compared to placebo for FP , BI 200 mg and MT (adj. mean difference, 95%CI, % difference): FP-1.64 (95% CI -2.18,-1.11),- 33%), 200 mg BI -0.85 (95% CI -1.40,-0.30), -17%), MT -0.74 (95% CI -1.31,-0.17), -15%) , 400 mg BI -0.51 (95% CI -1.09, 0.06), -10%), and 50 mg BI -0.4 (95% CI -1.01, 0.20), -8%). Inhibition of nasal IL-4, IL-5, IL-13, and eotaxin production was observed for BI, and FP. Reduction in absolute and % nasal eosinophils was observed for all treatments. Dose related reduction in ESC was seen only for BI but was not seen for FP, MT, or placebo. CONCLUSION: BI 671800 at a dose of 200 mg twice a day reduced TNSS vs. placebo over a 6 hour allergen challenge period; and at the end of the 6 hours showed a reduction in TH-2 inflammatory cytokines as well as a reduction in the number and % of nasal eosinophils compared to placebo