Search

RNA-binding proteins (RBPs), essential for skeletal muscle regeneration, cause muscle degeneration and neuromuscular disease when mutated. Why mutations in these ubiquitously expressed RBPs orchestrate complex tissue regeneration and direct cell fate decisions in skeletal muscle remains poorly understood. Single-cell RNA-sequencing of regenerating Mus musculus skeletal muscle reveals that RBP expression, including the expression of many neuromuscular disease-associated RBPs, is temporally regulated in skeletal muscle stem cells and correlates with specific stages of myogenic differentiation. By combining machine learning with RBP engagement scoring, we discovered that the neuromuscular disease-associated RBP Hnrnpa2b1 is a differentiation-specifying regulator of myogenesis that controls myogenic cell fate transitions during terminal differentiation in mice. The timing of RBP expression specifies cell fate transitions by providing post-transcriptional regulation of messenger RNAs that coordinate stem cell fate decisions during tissue regeneration.

Community testing programs focused on the unvaccinated population are being enacted in populations with mixed vaccination status to mitigate SARS-CoV-2 spread. Presumably, these policies assume that the unvaccinated are driving transmission, though it is not well understood how viral spread occurs in mixed-status populations. Here, we analyze a model of transmission in which a variable fraction of the population is vaccinated, with unvaccinated individuals proactively screened for infection. By exploring a range of transmission rates, vaccine effectiveness (VE) scenarios, and rates of prior infection, this analysis reveals principles of viral spread in communities of mixed vaccination status, with implications for screening policies. As vaccination rates increase, the proportion of transmission driven by the unvaccinated population decreases, such that most community spread is driven by breakthrough infections once vaccine coverage exceeds 55% (omicron) or 80% (delta), with additional variation dependent on waning or boosted VE. More broadly, the potential impacts of unvaccinated-only screening fall into three distinct parameter regions: (I) “flattening the curve” with little impact on cumulative infections, (II) effectively suppressing transmission, and (III) negligible impact because herd immunity is reached without screening. By evaluating a wide range of scenarios, this work finds broadly that effective mitigation of SARS-CoV-2 transmission by unvaccinated-only screening is highly dependent on vaccination rate, population-level immunity, screening compliance, and vaccine effectiveness against the current variant.

Stress granules (SGs) are cytoplasmic assemblies of RNA and protein that form when translation is repressed during the integrated stress response. SGs assemble from the combination of RNA–RNA, RNA–protein and protein–protein interactions between messenger ribonucleoprotein complexes (mRNPs). The protein adenosine deaminase acting on RNA 1 (ADAR1, also known as ADAR) recognizes and modifies double-stranded RNAs (dsRNAs) within cells to prevent an aberrant innate immune response. ADAR1 localizes to SGs, and since RNA–RNA interactions contribute to SG assembly and dsRNA induces SGs, we examined how ADAR1 affects SG formation. First, we demonstrate that ADAR1 depletion triggers SGs by allowing endogenous dsRNA to activate the integrated stress response through activation of PKR (also known as EIF2AK2) and translation repression. However, we also show that ADAR1 limits SG formation independently of translation inhibition. ADAR1 repression of SGs is independent of deaminase activity but is dependent on dsRNA-binding activity, suggesting a model where ADAR1 binding limits RNA–RNA and/or RNA–protein interactions necessary for recruitment to SGs. Given that ADAR1 expression is induced during viral infection, these findings have implications for the role of ADAR1 in the antiviral response. This article has an associated First Person interview with the first author of the paper.

Ribonucleoprotein (RNP) assemblies are ubiquitous in eukaryotic cells and have functions throughout RNA transcription, splicing, and stability. Of the RNA-binding proteins that form RNPs, TAR DNA-binding protein of 43 kD (TDP43) is of particular interest due to its essential nature and its association with disease. TDP43 plays critical roles in RNA metabolism, many of which require its recruitment to RNP granules such as stress granules, myo-granules, and neuronal transport granules. Moreover, the presence of cytoplasmic TDP43-positive inclusions is a pathological hallmark of several neurodegenerative diseases. Despite the pervasiveness of TDP43 aggregates, TDP43 mutations are exceedingly rare, suggesting that aggregation may be linked to dysregulation of TDP43 function. Oligomerization is a part of normal TDP43 function; thus, it is of interest to understand what triggers the irreversible aggregation that is seen in disease. Herein, we examine TDP43 functions, particularly in RNP granules, and the mechanisms which may explain pathological TDP43 aggregation.

Numerous membrane-less organelles composed of a combination of RNA and proteins, referred to as RNP granules, are observed in the nucleus and cytoplasm of eukaryotic cells, including stress granules, processing bodies, Cajal bodies, and nuclear speckles. An unresolved issue is how frequently RNA molecules are required for the maintenance of RNP granules in either the nucleus or cytosol. To address this issue, we degraded intracellular RNA in either the cytosol or the nucleus by the activation of RNase L and examined the impact of RNA loss on several RNP granules. Strikingly, we find the majority of RNP granules, including stress granules, processing bodies, Cajal bodies, nuclear speckles and the nucleolus are altered by the degradation of their RNA components. In contrast, super-enhancer complexes and TIS granules were largely unaffected by widespread intracellular RNA degradation. This highlights a critical and widespread role of RNA in the organization of many, but not all, RNP granules.SUMMARYDecker et al. examine the role of RNA in maintaining membrane-less organelles composed of RNA and protein by activating RNase L in the cytoplasm or nucleus. Degradation of RNA alters the majority of membrane-less organelles indicating that RNA plays a widespread role in their organization.

To rapidly respond and adapt to stresses, such as viral infections, cells have evolved several mechanisms, which include the activation of stress response pathways and the innate immune response. These stress responses result in the rapid inhibition of translation and condensation of stalled mRNAs, together with RNA-binding proteins and signalling components, into cytoplasmic biocondensates called stress granules. Increasing evidence suggests that stress granules contribute to antiviral defense and thus viruses need to evade these response pathways to propagate. In addition, the stress granule pathway is proposed to be dynamic and adaptable to specific stresses. We previously showed that Feline Calicivirus (FCV) impairs SGs assembly by cleaving the scaffolding protein G3BP1. We also observed that uninfected bystander cells assembled G3BP1-granules, suggesting a paracrine response trigged by the infection. We now present evidence that virus-free supernatant generated from infected cells can induce the formation of paracrine granules. They are different from canonical stress granules and exhibit specific kinetics of assembly-disassembly, protein and RNA composition and are linked to antiviral activity. We propose that this paracrine induction reflects a novel cellular defence mechanism to limit viral propagation and promote stress responses in bystander cells.Summary statementWe describe a novel type of paracrine induced RNA granules associated with viruses, highlighting how different stresses results in heterogeneous stress granule-like condensates with specific cellular functions.

To assist in the COVID-19 public health guidance on a college campus, daily composite wastewater samples were withdrawn at 20 manhole locations across the University of Colorado Boulder campus. Low-cost autosamplers were fabricated in-house to enable an economical approach to this distributed study. These sample stations operated from August 25th until November 23rd during the fall 2020 semester, with 1,512 samples collected. The concentration of SARS-CoV-2 in each sample was quantified through two comparative reverse transcription quantitative polymerase chain reactions (RT-qPCRs). These methods were distinct in the utilization of technical replicates and normalization to an endogenous control. (1) Higher temporal resolution compensates for supply chain or other constraints that prevent technical or biological replicates. (2) The data normalized by an endogenous control agreed with the raw concentration data, minimizing the utility of normalization. The raw wastewater concentration values reflected SARS-CoV-2 prevalence on campus as detected by clinical services. Overall, combining the low-cost composite sampler with a method that quantifies the SARS-CoV-2 signal within six hours enabled actionable and time-responsive data delivered to key stakeholders. With daily reporting of the findings, wastewater surveillance assisted in decision making during critical phases of the pandemic on campus, from detecting individual cases within populations ranging from 109 to 2,048 individuals to monitoring the success of on-campus interventions., Graphical abstract Tracking SARS-CoV-2 in on-campus wastewater informs and monitors public health decisions and actions. Image, graphical abstract

The transcriptional induction of interferon (IFN) genes is a key feature of the mammalian antiviral response that limits viral replication and dissemination. A hallmark of severe COVID-19 disease caused by SARS-CoV-2 is the low presence of IFN proteins in patient serum despite elevated levels of IFN-encoding mRNAs, indicative of post-transcriptional inhibition of IFN protein production. Here, we performed single-molecule RNA visualization to examine the expression and localization of host mRNAs during SARS-CoV-2 infection. Our data show that the biogenesis of type I and type III IFN mRNAs is inhibited at multiple steps during SARS-CoV-2 infection. First, translocation of the interferon regulatory factor 3 (IRF3) transcription factor to the nucleus is limited in response to SARS-CoV-2, indicating that SARS-CoV-2 inhibits RLR-MAVS signaling and thus weakens transcriptional induction of IFN genes. Second, we observed that IFN mRNAs primarily localize to the site of transcription in most SARS-CoV-2 infected cells, suggesting that SARS-CoV-2 either inhibits the release of IFN mRNAs from their sites of transcription and/or triggers decay of IFN mRNAs in the nucleus upon exiting the site of transcription. Lastly, nuclear-cytoplasmic transport of IFN mRNAs is inhibited during SARS-CoV-2 infection, which we propose is a consequence of widespread degradation of host cytoplasmic basal mRNAs in the early stages of SARS-CoV-2 replication by the SARS-CoV-2 Nsp1 protein, as well as the host antiviral endoribonuclease, RNase L. Importantly, IFN mRNAs can escape SARS-CoV-2-mediated degradation if they reach the cytoplasm, making rescue of mRNA export a viable means for promoting the immune response to SARS-CoV-2.

Aberrant translational repression is a feature of multiple neurodegenerative diseases. The association between disease-linked proteins and stress granules further implicates impaired stress responses in neurodegeneration. However, our knowledge of the proteins that evade translational repression is incomplete. It is also unclear whether disease-linked proteins influence the proteome under conditions of translational repression. To address these questions, a quantitative proteomics approach was used to identify proteins that evade stress-induced translational repression in arsenite-treated cells expressing either wild-type or amyotrophic lateral sclerosis (ALS)-linked mutant FUS. This study revealed hundreds of proteins that are actively synthesized during stress-induced translational repression, irrespective of FUS genotype. In addition to proteins involved in RNA- and protein-processing, proteins associated with neurodegenerative diseases such as ALS were also actively synthesized during stress. Protein synthesis under stress was largely unperturbed by mutant FUS, although several proteins were found to be differentially expressed between mutant and control cells. One protein in particular, COPBI, was downregulated in mutant FUS-expressing cells under stress. COPBI is the beta subunit of the coat protein I (COPI), which is involved in Golgi to endoplasmic reticulum (ER) retrograde transport. Further investigation revealed reduced levels of other COPI subunit proteins and defects in COPBI-relatedprocesses in cells expressing mutant FUS. Even in the absence of stress, COPBI localization was altered in primary and human stem cell-derived neurons expressing ALS-linked FUS variants. Our results suggest that Golgi to ER retrograde transport may be important under conditions of stress and is perturbed upon the expression of disease-linked proteins such as FUS.

Stress granules (SGs) are cytoplasmic RNA–protein aggregates formed in response to inhibition of translation initiation. SGs contribute to the stress response and are implicated in a variety of diseases, including cancer and some forms of neurodegeneration. Neurodegenerative diseases often involve chronic phosphorylation of eukaryotic initiation factor 2α (eIF2α), with deletions of eIF2α kinases or treatment with eIF2α kinase inhibitors being protective in some animal models of disease. However, how and why the integrated stress response (ISR) is activated in different forms of neurodegeneration remains unclear. Because neuroinflammation is common to many neurodegenerative diseases, we hypothesized that inflammatory factors contribute to ISR activation in a cell-nonautonomous manner. Using fluorescence microscopy and immunoblotting, we show here that the endogenously produced product of inflammation, 15-deoxy-Δ12,14-prostaglandin J2 (15-d-PGJ2), triggers eIF2α phosphorylation, thereby activating the ISR, repressing bulk translation, and triggering SG formation. Our findings define a mechanism by which inflammation activates the ISR in a cell-nonautonomous manner and suggest that inhibition of 15-d-PGJ2 production might be a useful therapeutic strategy in some neuroinflammatory contexts.

Ribonucleoprotein (RNP) granules are non-membrane-bound organelles that have critical roles in the stress response1,2, maternal messenger RNA storage3, synaptic plasticity4, tumour progression5,6 and neurodegeneration7-9. However, the dynamics of their mRNA components within and near the granule surface remain poorly characterized, particularly in the context and timing of mRNAs exiting translation. Herein, we used multicolour single-molecule tracking to quantify the precise timing and kinetics of single mRNAs as they exit translation and enter RNP granules during stress. We observed single mRNAs interacting with stress granules and P-bodies, with mRNAs moving bidirectionally between them. Although translating mRNAs only interact with RNP granules dynamically, non-translating mRNAs can form stable, and sometimes rigid, associations with RNP granules with stability increasing with both mRNA length and granule size. Live and fixed cell imaging demonstrated that mRNAs can extend beyond the protein surface of a stress granule, which may facilitate interactions between RNP granules. Thus, the recruitment of mRNPs to RNP granules involves dynamic, stable and extended interactions affected by translation status, mRNA length and granule size that collectively regulate RNP granule dynamics.

A central problem in the COVID-19 pandemic is that there is not enough testing to prevent infectious spread of SARS-CoV-2, causing surges and lockdowns with human and economic toll. Molecular tests that detect viral RNAs or antigens will be unable to rise to this challenge unless testing capacity increases by at least an order of magnitude while decreasing turnaround times. Here, we evaluate an alternative strategy based on the monitoring of olfactory dysfunction, a symptom identified in 76–83% of SARS-CoV-2 infections—including those with no other symptoms—when a standardized olfaction test is used. We model how screening for olfactory dysfunction, with reflexive molecular tests, could be beneficial in reducing community spread of SARS-CoV-2 by varying testing frequency and the prevalence, duration, and onset time of olfactory dysfunction. We find that monitoring olfactory dysfunction could reduce spread via regular screening, and could reduce risk when used at point-of-entry for single-day events. In light of these estimated impacts, and because olfactory tests can be mass produced at low cost and self-administered, we suggest that screening for olfactory dysfunction could be a high impact and cost-effective method for broad COVID-19 screening and surveillance., A central problem in the COVID-19 pandemic is that there is not enough testing to prevent infectious spread of SARS-CoV-2, causing surges and lockdowns with human and economic toll. Here, the authors evaluate an alternative strategy based on the monitoring of olfactory dysfunction with a mathematical model.

RNase L–mediated shutoff of nuclear mRNA export limits viral protein synthesis and regulates antiviral cytokine production., RNase L is widely thought to limit viral protein synthesis by cleaving host rRNA and viral mRNA, resulting in translation arrest and viral mRNA degradation. Here, we show that the mRNAs of dengue virus and influenza A virus largely escape RNase L–mediated mRNA decay, and this permits viral protein production. However, activation of RNase L arrests nuclear mRNA export, which strongly inhibits influenza A virus protein synthesis and reduces cytokine production. The heterogeneous and temporal nature of the mRNA export block in individual cells permits sufficient production of antiviral cytokines from transcriptionally induced host mRNAs. This defines RNase L–mediated arrest of mRNA export as a key antiviral shutoff and cytokine regulatory pathway.

Significance We analyzed data from saliva-based COVID-19 screening deployed on the University of Colorado Boulder campus. Our dataset is unique in that all SARS-CoV-2−positive individuals reported no symptoms at the time of saliva collection, and therefore were infected but asymptomatic or presymptomatic. We found that 1) the distribution of viral loads observed in our asymptomatic college population was indistinguishable from what has been reported in hospitalized populations; 2) regardless of symptomatic status, approximately 50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the infection; and 3) just 2% of infected individuals carry 90% of the virions circulating within communities, serving as viral “supercarriers” and likely also superspreaders., We analyze data from the fall 2020 pandemic response efforts at the University of Colorado Boulder, where more than 72,500 saliva samples were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using qRT-PCR. All samples were collected from individuals who reported no symptoms associated with COVID-19 on the day of collection. From these, 1,405 positive cases were identified. The distribution of viral loads within these asymptomatic individuals was indistinguishable from what has been previously observed in symptomatic individuals. Regardless of symptomatic status, ∼50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the disease, based on having low viral loads in a range from which live virus has rarely been isolated. We find that, at any given time, just 2% of individuals carry 90% of the virions circulating within communities, serving as viral “supercarriers” and possibly also superspreaders.

Stress granules (SGs) are ribonucleoprotein (RNP) assemblies that form in eukaryotic cells as a result of limited translation in response to stress. SGs form during viral infection and are thought to promote the antiviral response because many viruses encode inhibitors of SG assembly. However, the antiviral endoribonuclease RNase L also alters SG formation, whereby only small punctate SG-like bodies that we term RNase L-dependent bodies (RLBs) form during RNase L activation. How RLBs relate to SGs and their mode of biogenesis is unknown. Herein, using immunofluorescence, live-cell imaging, and MS-based analyses, we demonstrate that RLBs represent a unique RNP granule with a protein and RNA composition distinct from that of SGs in response to dsRNA lipofection in human cells. We found that RLBs are also generated independently of SGs and the canonical dsRNA-induced SG biogenesis pathway, because RLBs did not require protein kinase R, phosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eIF2α), the SG assembly G3BP paralogs, or release of mRNAs from ribosomes via translation elongation. Unlike the transient interactions between SGs and P-bodies, RLBs and P-bodies extensively and stably interacted. However, despite both RLBs and P-bodies exhibiting liquid-like properties, they remained distinct condensates. Taken together, these observations reveal that RNase L promotes the formation of a unique RNP complex that may have roles during the RNase L-mediated antiviral response.

A key feature of the mammalian innate immune response to viral infection is the transcriptional induction of interferon (IFN) genes, which encode for secreted proteins that prime the antiviral response and limit viral replication and dissemination. A hallmark of severe COVID-19 disease caused by SARS-CoV-2 is the low presence of IFN proteins in patient serum despite elevated levels of IFN-encoding mRNAs, indicative of post-transcriptional inhibition of IFN protein production. Herein, we show SARS-CoV-2 infection limits type I and type III IFN biogenesis by preventing the release of mRNA from their sites of transcription and/or triggering their nuclear degradation. In addition, SARS-CoV-2 infection inhibits nuclear-cytoplasmic transport of IFN mRNAs as a consequence of widespread cytosolic mRNA degradation mediated by both activation of the host antiviral endoribonuclease, RNase L, and by the SARS-CoV-2 protein, Nsp1. These findings argue that inhibition of host and/or viral Nsp1-mediated mRNA decay, as well as IFN treatments, may reduce viral-associated pathogenesis by promoting the innate immune response.

The presence of the m

RNA-binding proteins (RBPs) are essential for skeletal muscle regeneration and RBP dysfunction causes muscle degeneration and neuromuscular disease. How ubiquitously expressed RBPs orchestrate complex tissue regeneration and direct cell fate decisions in skeletal muscle remains poorly understood. Single cell RNA-sequencing of regenerating skeletal muscle reveals that RBP expression, including numerous neuromuscular disease-associated RBPs, is temporally regulated in skeletal muscle stem cells and correlates to stages of myogenic differentiation. By combining machine learning with RBP engagement scoring, we discover that the neuromuscular disease associated RBP Hnrnpa2b1 is a differentiation-specifying regulator of myogenesis controlling myogenic cell fate transitions during terminal differentiation. The timing of RBP expression specifies cell fate transitions by providing a layer of post-transcriptional regulation needed to coordinate stem cell fate decisions during complex tissue regeneration.

In 2019-2020, the COVID-19 pandemic spread to over 200 countries in less than six months. To understand the basis of this aggressive spread, it is essential to determine the transmission rate and define the factors that increase the risk of transmission. One complication is the large fraction of asymptomatic cases, particularly in young populations: these individuals have viral loads indistinguishable from symptomatic people and do transmit the SARS-CoV-2 virus, but they often go undetected. As university students living in residence halls commonly share a small living space with roommates, some schools established regular, high density testing programs to mitigate on-campus spread. In this study, we analyzed longitudinal testing data of residence hall students at the University of Colorado Boulder. We observed that students in single rooms were infected at a lower rate than students in multiple occupancy rooms. However, this was not due to high rates of transmission between roommates, which only occurred approximately 20% of the time. Since these cases were usually asymptomatic at the time of diagnosis, this provides further evidence for asymptomatic transmission. Notably, individuals who likely transmitted to their roommates had an average viral load ∼6.5 times higher than individuals who did not. Although students were moved to separate isolation rooms after diagnosis, there was no difference in time to isolation between these cases with or without transmission. This analysis argues that inter-roommate transmission occurs in a minority of cases in university residence halls and provides strong correlative evidence that viral load can be proportional to the probability of transmission.

Tau aggregates contribute to neurodegenerative diseases including frontotemporal dementia and Alzheimer’s disease (AD). Although RNA promotes tau aggregation in vitro, whether tau aggregates in cells contain RNA is unknown. We demonstrate in cell culture and mouse brains that both cytosolic and nuclear tau aggregates contain RNA, with enrichment for snRNAs and snoRNAs. Nuclear tau aggregates colocalize with and alter the composition, dynamics, and organization of nuclear speckles, which are membraneless organelles involved in pre-mRNA splicing. Moreover, several nuclear speckle components, including SRRM2, mislocalize to cytosolic tau aggregates in cells, mouse brains, and patient brains with AD, frontotemporal dementia (FTD), and corticobasal degeneration (CBD). Consistent with these alterations we observe the presence of tau aggregates is sufficient to alter pre-mRNA splicing. This work identifies tau alteration of nuclear speckles as a feature of tau aggregation that may contribute to the pathology of tau aggregates.

A key feature of the mammalian antiviral response is the transcriptional induction of interferon (IFN) genes, which encode for secreted proteins that prime the antiviral response to limit viral replication and dissemination. A hallmark of severe COVID-19 disease caused by SARS-CoV-2 is the low presence of IFNs in patient serum despite elevated levels of IFN-encoding mRNAs, indicative of post-transcriptional inhibition of IFN protein production. Herein, we show SARS-CoV-2 infection limits type I and type III IFN biogenesis by preventing the release of mRNA from their sites of transcription and/or triggering their nuclear degradation. Additionally, SARS-CoV-2 infection inhibits nuclear-cytoplasmic transport of IFN mRNAs as a consequence of widespread cytosolic mRNA degradation mediated by both the host antiviral endoribonuclease, RNase L, and by the SARS-CoV-2 protein, Nsp1. Thus, inhibition of host and/or viral Nsp1-mediated mRNA decay, as well as IFN treatments, may reduce viral-associated pathogenesis by promoting the innate immune response. Funding Information: Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number F32AI145112 (J.M.B), funds from HHMI (Roy Parker), and support provided by the Office of the Vice President for Research and the Dept. of Microbiology, Immunology and Pathology at Colorado State University (Rushika Perera). Declaration of Interests: Roy Parker is a founder and consultant of Faze Medicines.

A central problem in the COVID-19 pandemic is that there is not enough testing to prevent infectious spread of SARS-CoV-2, causing surges and lockdowns with human and economic toll. Molecular tests that detect viral RNAs or antigens will be unable to rise to this challenge unless testing capacity increases by at least an order of magnitude while decreasing turnaround times. Here, we evaluate an alternative strategy based on the monitoring of olfactory dysfunction, a symptom identified in 76-83% of SARS-CoV-2 infections—including those with no other symptoms—when a standardized olfaction test is used. We model how screening for olfactory dysfunction, with reflexive molecular tests, could be beneficial in reducing community spread of SARS-CoV-2 by varying testing frequency and the prevalence, duration, and onset time of olfactory dysfunction. We find that monitoring olfactory dysfunction could reduce spread via regular screening, and could reduce risk when used at point-of-entry for single-day events. In light of these estimated impacts, and because olfactory tests can be mass produced at low cost and self-administered, we suggest that screening for olfactory dysfunction could be a high impact and cost-effective method for broad COVID-19 screening and surveillance.

Test sensitivity is secondary to frequency and turnaround time for COVID-19 screening., The COVID-19 pandemic has created a public health crisis. Because SARS-CoV-2 can spread from individuals with presymptomatic, symptomatic, and asymptomatic infections, the reopening of societies and the control of virus spread will be facilitated by robust population screening, for which virus testing will often be central. After infection, individuals undergo a period of incubation during which viral titers are too low to detect, followed by exponential viral growth, leading to peak viral load and infectiousness and ending with declining titers and clearance. Given the pattern of viral load kinetics, we model the effectiveness of repeated population screening considering test sensitivities, frequency, and sample-to-answer reporting time. These results demonstrate that effective screening depends largely on frequency of testing and speed of reporting and is only marginally improved by high test sensitivity. We therefore conclude that screening should prioritize accessibility, frequency, and sample-to-answer time; analytical limits of detection should be secondary.

Serpentoviruses are an emerging group of nidoviruses known to cause respiratory disease in snakes and have been associated with disease in other non-avian reptile species (lizards and turtles). This study describes multiple episodes of respiratory disease-associated mortalities in a collection of juvenile veiled chameleons (Chamaeleo calyptratus). Histopathologic lesions included rhinitis and interstitial pneumonia with epithelial proliferation and abundant mucus. Metagenomic sequencing detected coinfection with two novel serpentoviruses and a novel orthoreovirus. Veiled chameleon serpentoviruses are most closely related to serpentoviruses identified in snakes, lizards, and turtles (approximately 40–50% nucleotide and amino acid identity of ORF1b). Veiled chameleon orthoreovirus is most closely related to reptilian orthoreoviruses identified in snakes (approximately 80–90% nucleotide and amino acid identity of the RNA-dependent RNA polymerase). A high prevalence of serpentovirus infection (>80%) was found in clinically healthy subadult and adult veiled chameleons, suggesting the potential for chronic subclinical carriers. Juvenile veiled chameleons typically exhibited a more rapid progression compared to subadults and adults, indicating a possible age association with morbidity and mortality. This is the first description of a serpentovirus infection in any chameleon species. A causal relationship between serpentovirus infection and respiratory disease in chameleons is suspected. The significance of orthoreovirus coinfection remains unknown.

Stress granules (SGs) are stress-induced RNA–protein assemblies formed from a complex transcriptome of untranslating ribonucleoproteins (RNPs). Although RNAs can be either enriched or depleted from SGs, the rules that dictate RNA partitioning into SGs are unknown. We demonstrate that the SG-enriched NORAD RNA is sufficient to enrich a reporter RNA within SGs through the combined effects of multiple elements. Moreover, artificial tethering of G3BP1, TIA1, or FMRP can target mRNAs into SGs in a dose-dependent manner with numerous interactions required for efficient SG partitioning, which suggests individual protein interactions have small effects on the SG partitioning of mRNPs. This is supported by the observation that the SG transcriptome is largely unchanged in cell lines lacking the abundant SG RNA-binding proteins G3BP1 and G3BP2. We suggest the targeting of RNPs into SGs is due to a summation of potential RNA–protein, protein–protein, and RNA–RNA interactions with no single interaction dominating RNP recruitment into SGs.

Rethinking Covid-19 Test Sensitivity As Covid-19 cases accelerate or plateau around the world, we urgently need a point-of-care test that is inexpensive enough to use frequently, even if it lacks h...

Here, we develop a simple molecular test for SARS-CoV-2 in saliva based on reverse transcription loop-mediated isothermal amplification (RT-LAMP). The test has two steps: 1) heat saliva with a stabilization solution, and 2) detect virus by incubating with a primer/enzyme mix. After incubation, saliva samples containing the SARS-CoV-2 genome turn bright yellow. Because this test is pH dependent, it can react falsely to some naturally acidic saliva samples. We report unique saliva stabilization protocols that rendered 295 healthy saliva samples compatible with the test, producing zero false positives. We also evaluated the test on 278 saliva samples from individuals who were infected with SARS-CoV-2 but had no symptoms at the time of saliva collection, and from 54 matched pairs of saliva and anterior nasal samples from infected individuals. The Saliva TwoStep test described herein identified infections with 94% sensitivity and >99% specificity in individuals with sub-clinical (asymptomatic or pre-symptomatic) infections.

The COVID-19 pandemic has created a public health crisis. Because SARS-CoV-2 can spread from individuals with pre-symptomatic, symptomatic, and asymptomatic infections, the re-opening of societies and the control of virus spread will be facilitated by robust surveillance, for which virus testing will often be central. After infection, individuals undergo a period of incubation during which viral titers are usually too low to detect, followed by an exponential growth of virus, leading to a peak viral load and infectiousness, and ending with declining viral levels and clearance. Given the pattern of viral load kinetics, we model surveillance effectiveness considering test sensitivities, frequency, and sample-to-answer reporting time. These results demonstrate that effective surveillance, including time to first detection and outbreak control, depends largely on frequency of testing and the speed of reporting, and is only marginally improved by high test sensitivity. We therefore conclude that surveillance should prioritize accessibility, frequency, and sample-to-answer time; analytical limits of detection should be secondary.

It is now clear that cells form a wide collection of large RNA-protein assemblies, referred to as RNP granules. RNP granules exist in bacterial cells and can be found in both the cytosol and nucleus of eukaryotic cells. Recent approaches have begun to define the RNA and protein composition of a number of RNP granules. Herein, we review the composition and assembly of RNP granules, as well as how RNPs are targeted to RNP granules using stress granules and P-bodies as model systems. Taken together, these reveal that RNP granules form through the summative effects of a combination of protein-protein, protein-RNA, and RNA-RNA interactions. Similarly, the partitioning of individual RNPs into stress granules is determined by the combinatorial effects of multiple elements. Thus, RNP granules are assemblies generally dominated by combinatorial effects, thereby providing rich opportunities for biological regulation.

INTRODUCTION The cytoplasm contains an unconventional class of organelles that concentrate specific factors and resources without a limiting membrane. These membraneless organelles include ribonucleoprotein (RNP) granules such as processing bodies (P-bodies, or PBs) and stress granules. PBs and stress granules are composed of nontranslating messenger RNAs (mRNAs) and associated proteins and are thought to provide discrete biochemical environments for regulating the translation and/or degradation of mRNA. In contrast to membrane-bound organelles, very little is known about what extrinsic and intrinsic factors regulate the fusion and fission of membrane-less organelles. Recently, an unexpected role for the endoplasmic reticulum (ER) has been observed in regulating the biogenesis of other membrane-bound organelles at contact sites where the two organelles are tethered and closely apposed. ER contact sites can allow the direct exchange of macromolecules and serve as a platform for the recruitment of machineries that regulate organelle biogenesis, division, and trafficking. Here, we found that ER contact sites can also regulate the biogenesis and fission of two types of membraneless organelles, PBs and stress granules. RATIONALE To determine the extent to which PBs, a conserved cytoplasmic membraneless organelle, are tethered to the ER in animal cells, we used live-cell fluorescence microscopy to simultaneously track the spatiotemporal dynamics of the ER and PBs. To overcome the diffraction limits associated with light microscopy, we designed a reversible ER-PB contact assay using probes attached to the ER and PBs that emit a high-intensity fluorescence signal when the probes are close enough to dimerize. Because ER morphology and RNP granule biogenesis are tightly linked to mRNA translation, we systematically evaluated the relationships between ER morphology, RNP granule biogenesis, and mRNA translation by assessing endogenous PB numbers in response to altering ER shape and translational capacity and to the induction of cytosolic and ER stress. Because PBs and stress granules are dynamic organelles that undergo fission and fusion reactions akin to membrane-bound organelles, we used live-cell fluorescence microscopy to score the spatiotemporal relationship between the position of RNP granule division and contact sites with ER tubules. RESULTS Using multiple measures, we found that a population of PBs were tethered to the ER in human cells. ER shape exerted profound effects on PB numbers and PB-ER contact. Conditions that promoted expansion of peripheral ER tubules and a reduction in peripheral ER cisternae increased PB numbers and ER-PB contact. Conversely, conditions that promoted an expansion of ER cisternae dramatically decreased PB numbers. The effect of ER shape on PB abundance was likely a reflection of the relative translational capacity of the ER domains. Owing to differences in ribosome density, smooth ER tubules are presumed to have a lower translational capacity than rough ER cisternae. Conditions that locally enhanced the translational capacity of the ER by increasing ER cisternae, such as ER stress, also reduced the number of PBs. Conversely, conditions that globally inhibited mRNA translation (NaAsO2 and puromycin) suppressed the effects of ER shape on PB abundance. Thus, ER contact sites affected the proliferation of PBs under basal and translationally repressed conditions. Furthermore, ER contact sites also affected the mysterious PB fission process. Live-cell imaging revealed that dynamic ER tubules define the position where PB and stress granule division occurs. These data mirror the spatiotemporal role of ER tubule contact domains that drive the constriction and division of membrane-bound organelles like endosomes and mitochondria. CONCLUSION Here, we found that the ER contains contact site domains that are capable of tethering both membraneless and membrane-bound organelles. ER structure and translational capacity has effects on PB biogenesis. Furthermore, the fission of cytoplasmic RNP granules appears to represent an active process that can be driven by ER contact sites, analogous to the division of membrane-bound organelles.

Viral infections impose major stress on the host cell. In response, stress pathways can rapidly deploy defence mechanisms by shutting off the protein synthesis machinery and triggering the accumulation of mRNAs into stress granules to limit the use of energy and nutrients. Because this threatens viral gene expression, viruses need to evade these pathways to propagate. Human norovirus is responsible for gastroenteritis outbreaks worldwide. Here we examined how norovirus interacts with the eIF2α signaling axis controlling translation and stress granules. While norovirus infection represses host cell translation, our mechanistic analyses revealed that eIF2α signaling mediated by the stress kinase GCN2 is uncoupled from translational stalling. Moreover, infection results in a redistribution of the RNA-binding protein G3BP1 to replication complexes and remodelling of its interacting partners, allowing the avoidance from canonical stress granules. These results define novel strategies by which norovirus undergo efficient replication whilst avoiding the host stress response and manipulating the G3BP1 interactome., Author summary Viruses have evolved elegant strategies to evade host responses that restrict viral propagation by targeting the protein synthesis machinery and stress granules, which are membrane-less RNA granules with antiviral properties. Previous studies have unravelled how viruses, including norovirus the leading cause of gastroenteritis, regulate the activity of translation factors to affect the antiviral response. Furthermore, stress granules evasion strategies have been linked to targeting the scaffolding protein G3BP1. Here we dissect how murine norovirus, the main model for norovirus, evades the cellular stress responses. Our work challenges the dogma that translational control during infection is mainly mediated by eIF2α and demonstrate that norovirus evades this stress pathway. We further show that norovirus evades the stress granule response in a novel way by isolating and characterising the G3BP1 interactome for the first time in the context of a viral infection. We conclude that norovirus infection results in a redistribution of G3BP1 and its cellular partners to replication complexes, thereby preventing the assembly of stress granules. Overall, we define a novel evasion strategy by which norovirus escapes stress granule formation by rewiring the G3BP1 interactome.

Moon et al., demonstrate a new role for ribosome-associated quality control (RQC) factors in mediating mRNA release from ribosomes during stress to promote their localization to stress granules., Stress granules are dynamic assemblies of proteins and nontranslating RNAs that form when translation is inhibited in response to diverse stresses. Defects in ubiquitin–proteasome system factors including valosin-containing protein (VCP) and the proteasome impact the kinetics of stress granule induction and dissolution as well as being implicated in neuropathogenesis. However, the impacts of dysregulated proteostasis on mRNA regulation and stress granules are not well understood. Using single mRNA imaging, we discovered ribosomes stall on some mRNAs during arsenite stress, and the release of transcripts from stalled ribosomes for their partitioning into stress granules requires the activities of VCP, components of the ribosome-associated quality control (RQC) complex, and the proteasome. This is an unexpected contribution of the RQC system in releasing mRNAs from translation under stress, thus identifying a new type of stress-activated RQC (saRQC) distinct from canonical RQC pathways in mRNA substrates, cellular context, and mRNA fate.

Stress granules (SGs) are membraneless organelles that form in eukaryotic cells after stress exposure [1] (reviewed in [2-4]). Following translation inhibition, polysome disassembly releases 48S preinitiation complexes (PICs). mRNA, PICs, and other proteins coalesce in SG cores [1, 5-7]. SG cores recruit a dynamic shell, whose properties are dominated by weak interactions between proteins and RNAs [8-10]. The structure and assembly of SGs and how different components contribute to their formation are not fully understood. Using super-resolution and expansion microscopy, we find that the SG component UBAP2L [11, 12] and the core protein G3BP1 [5, 11-13] occupy different domains inside SGs. UBAP2L displays typical properties of a core protein, indicating that cores of different compositions coexist inside the same granule. Consistent with a role as a core protein, UBAP2L is required for SG assembly in several stress conditions. Our reverse genetic and cell biology experiments suggest that UBAP2L forms granules independent of G3BP1 and 2 but does not interfere with stress-induced translational inhibition. We propose a model in which UBAP2L is an essential SG nucleator that acts upstream of G3BP1 and 2 and facilitates G3BP1 core formation and SG assembly and growth.

A dominant histopathological feature in neuromuscular diseases, including amyotrophic lateral sclerosis and inclusion body myopathy, is cytoplasmic aggregation of the RNA-binding protein TDP-43. Although rare mutations in TARDBP-the gene that encodes TDP-43-that lead to protein misfolding often cause protein aggregation, most patients do not have any mutations in TARDBP. Therefore, aggregates of wild-type TDP-43 arise in most patients by an unknown mechanism. Here we show that TDP-43 is an essential protein for normal skeletal muscle formation that unexpectedly forms cytoplasmic, amyloid-like oligomeric assemblies, which we call myo-granules, during regeneration of skeletal muscle in mice and humans. Myo-granules bind to mRNAs that encode sarcomeric proteins and are cleared as myofibres mature. Although myo-granules occur during normal skeletal-muscle regeneration, myo-granules can seed TDP-43 amyloid fibrils in vitro and are increased in a mouse model of inclusion body myopathy. Therefore, increased assembly or decreased clearance of functionally normal myo-granules could be the source of cytoplasmic TDP-43 aggregates that commonly occur in neuromuscular disease.

A key site of translation control is the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α), which reduces the rate of GDP to GTP exchange by eIF2B, leading to altered translation. The extent of eIF2α phosphorylation within neurons can alter synaptic plasticity. Phosphorylation of eIF2α is triggered by four stress-responsive kinases, and as such eIF2α is often phosphorylated during neurological perturbations or disease. Moreover, in some cases decreasing eIF2α phosphorylation mitigates neurodegeneration, suggesting that this could be a therapeutic target. Mutations in the γ subunit of eIF2, the guanine exchange factor eIF2B, an eIF2α phosphatase, or in two eIF2α kinases can cause disease in humans, demonstrating the importance of proper regulation of eIF2α phosphorylation for health.

SUMMARY Eukaryotic cells contain large RNA-protein assemblies referred to as RNP granules, whose assembly is promoted by both traditional protein interactions and intrinsically disordered protein domains. Using RNP granules as an example, we provide evidence for an assembly mechanism of large cellular structures wherein specific protein-protein or protein-RNA interactions act together with promiscuous interactions of intrinsically disordered regions (IDRs). This synergistic assembly mechanism illuminates RNP granule assembly and explains why many components of RNP granules, and other large dynamic assemblies, contain IDRs linked to specific protein-protein or protein-RNA interaction modules. We suggest assemblies based on combinations of specific interactions and promiscuous IDRs are common features of eukaryotic cells., In Brief Many RNA-binding proteins contain disordered regions. Protter et al. find that these regions interact nonspecifically with other proteins, which can disrupt phase separation in vitro. They also find that promiscuous interactions synergize with specific interactions to promote phase separation and formation of higher-order structures, like RNP granules.

Roy Parker, who died in January 2017, was one of the first researchers to study foster care and adoption and was Chair of the British Association for Adoption and Fostering (BAAF – now CoramBAAF) for six years from 1980. One of his last projects was a historical review of the concept of severe and multiple disadvantage and responses to it. At a time when 13.5 million people, including 30% of the UK’s children, are living in poverty (Joseph Rowntree Foundation, 2016) we publish it to mark his enormous contribution to the welfare of disadvantaged children and families.

The MS2-MCP system enables researchers to image multiple steps of the mRNA life cycle with high temporal and spatial resolution. However, for short-lived mRNAs, the tight binding of the MS2 coat protein (MCP) to the MS2 binding sites (MBS) protects the RNA from being efficiently degraded, and this confounds the study of mRNA regulation. Here, we describe a reporter system (MBSV6) with reduced affinity for the MCP, which allows mRNA degradation while preserving single-molecule detection determined by single-molecule FISH (smFISH) or live imaging. Constitutive mRNAs (MDN1 and DOA1) and highly-regulated mRNAs (GAL1 and ASH1) endogenously tagged with MBSV6 in Saccharomyces cerevisiae degrade normally. As a result, short-lived mRNAs were imaged throughout their complete life cycle. The MBSV6 reporter revealed that, in contrast to previous findings, coordinated recruitment of mRNAs at specialized structures such as P-bodies during stress did not occur, and mRNA degradation was heterogeneously distributed in the cytoplasm.

Purpose The purpose of this paper is to chart the history of personal social services for children and families in the UK and examine the factors that have influenced it. Special attention is given to changing perceptions of rights, the impact of scandals and the contribution of child development research. Design/methodology/approach Analysis of historical documents and research reports using four methods: a timeline of milestones, demarcation of distinct developmental periods, trends in policy and practice and comparisons of children’s needs and experiences at different times. Findings The evolution of services has not been linear. In policy, there have been reform and retrenchment, amalgamation and differentiation. Practice has been shaped by the emergence of new problems and the disappearance of old ones as well as by legislation, extreme events, research and finance, all occurring in specific political, moral and economic contexts. Originality/value An analysis of developments in children’s services in their political, economic, moral and research contexts.

The proper regulation of mRNA processing, localization, translation, and degradation occurs on mRNPs. However, the global principles of mRNP organization are poorly understood. We utilize the limited, but existing, information available to present a speculative synthesis of mRNP organization with the following key points. First, mRNPs form a compacted structure due to the inherent folding of RNA. Second, the ribosome is the principal mechanism by which mRNA regions are partially decompacted. Third, mRNPs are 50%–80% protein by weight, consistent with proteins modulating mRNP organization, but also suggesting the majority of mRNA sequences are not directly interacting with RNA-binding proteins. Finally, the ratio of mRNA-binding proteins to mRNAs is higher in the nucleus to allow effective RNA processing and limit the potential for nuclear RNA based aggregation. This synthesis of mRNP understanding provides a model for mRNP biogenesis, structure, and regulation with multiple implications.

The eukaryotic cytosol contains multiple RNP granules, including P-bodies and stress granules. Three different methods have been used to describe the transcriptome of stress granules or P-bodies, but how these methods compare and how RNA partitioning occurs between P-bodies and stress granules have not been addressed. Here, we compare the analysis of the stress granule transcriptome based on differential centrifugation with and without subsequent stress granule immunopurification. We find that while differential centrifugation alone gives a first approximation of the stress granule transcriptome, this methodology contains nonspecific transcripts that play a confounding role in the interpretation of results. We also immunopurify and compare the RNAs in stress granules and P-bodies under arsenite stress and compare those results to those for the P-body transcriptome described under nonstress conditions. We find that the P-body transcriptome is dominated by poorly translated mRNAs under nonstress conditions, but during arsenite stress, when translation is globally repressed, the P-body transcriptome is very similar to the stress granule transcriptome. This suggests that translation is a dominant factor in targeting mRNAs into both P-bodies and stress granules, and during stress, when most mRNAs are untranslated, the composition of P-bodies reflects this broader translation repression.

RNA viruses are a major source of emerging and re-emerging infectious diseases around the world. We developed a method to identify RNA viruses that is based on the fact that RNA viruses produce double-stranded RNA (dsRNA) while replicating. Purifying and sequencing dsRNA from the total RNA isolated from infected tissue allowed us to recover dsRNA virus sequences and replicated sequences from single-stranded RNA (ssRNA) viruses. We refer to this approach as dsRNA-Seq. By assembling dsRNA sequences into contigs we identified full length or partial RNA viral genomes of varying genome types infecting mammalian culture samples, identified a known viral disease agent in laboratory infected mice, and successfully detected naturally occurring RNA viral infections in reptiles. Here, we show that dsRNA-Seq is a preferable method for identifying viruses in organisms that don&rsquo, t have sequenced genomes and/or commercially available rRNA depletion reagents. In addition, a significant advantage of this method is the ability to identify replicated viral sequences of ssRNA viruses, which is useful for distinguishing infectious viral agents from potential noninfectious viral particles or contaminants.