Hypoxia and inflammation induce synergistic transcriptome turnover in macrophages.

Macrophages are effector immune cells that experience substantial changes to oxygenation when transiting through tissues, especially when entering tumors or infected wounds. How hypoxia alters gene expression and macrophage effector function at the post-transcriptional level remains poorly understood. Here, we use TimeLapse-seq to measure how inflammatory activation modifies the hypoxic response in primary macrophages. Nucleoside recoding sequencing allows the derivation of steady-state transcript levels, degradation rates, and transcriptional synthesis rates from the same dataset. We find that hypoxia produces distinct responses from resting and inflammatory macrophages. Hypoxia induces destabilization of mRNA transcripts, though inflammatory macrophages substantially increase mRNA degradation compared to resting macrophages. Increased RNA turnover results in the upregulation of ribosomal protein genes and downregulation of extracellular matrix components in inflammatory macrophages. Pathways regulated by mRNA decay in vitro are differentially regulated in tumor-associated macrophages implying that mixed stimuli could induce post-transcriptional regulation of macrophage function in solid tumors.

Regulation of translation in response to iron deficiency in human cells.

Protein synthesis is a highly energy-consuming process that is downregulated in response to many environmental stresses or adverse conditions. Studies in the yeast Saccharomyces cerevisiae have shown that bulk translation is inhibited during adaptation to iron deficiency, which is consistent with its requirement for ribosome biogenesis and recycling. Although iron deficiency anemia is the most common human nutritional disorder, how iron modulates translation in mammals is poorly understood. Studies during erythropoiesis have shown that iron bioavailability is coordinated with globin synthesis via bulk translation regulation. However, little is known about the control of translation during iron limitation in other tissues. Here, we investigated how iron depletion affects protein synthesis in human osteosarcoma U-2 OS cells. By adding an extracellular iron chelator, we observed that iron deficiency limits cell proliferation, induces autophagy, and decreases the global rate of protein synthesis. Analysis of specific molecular markers indicates that the inhibition of bulk translation upon iron limitation occurs through the eukaryotic initiation factor eIF2α and mechanistic target of rapamycin (mTOR) pathways. In contrast to other environmental and nutritional stresses, iron depletion does not trigger the assembly of messenger ribonucleoprotein stress granules, which typically form upon polysome disassembly.

DDX6 modulates P-body and stress granule assembly, composition, and docking.

Stress granules and P-bodies are ribonucleoprotein (RNP) granules that accumulate during the stress response due to the condensation of untranslating mRNPs. Stress granules form in part by intermolecular RNA-RNA interactions and can be limited by components of the RNA chaperone network, which inhibits RNA-driven aggregation. Herein, we demonstrate that the DEAD-box helicase DDX6, a P-body component, can also limit the formation of stress granules, independent of the formation of P-bodies. In an ATPase, RNA-binding dependent manner, DDX6 limits the partitioning of itself and other RNPs into stress granules. When P-bodies are limited, proteins that normally partition between stress granules and P-bodies show increased accumulation within stress granules. Moreover, we show that loss of DDX6, 4E-T, and DCP1A increases P-body docking with stress granules, which depends on CNOT1 and PAT1B. Taken together, these observations identify a new role for DDX6 in limiting stress granules and demonstrate that P-body components can influence stress granule composition and docking with P-bodies.

Identification of small molecule inhibitors of G3BP-driven stress granule formation.

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the RNA-binding proteins G3BP1/2. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting the co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress and dissolve pre-existing stress granules. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent powerful tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

Identification of small molecule inhibitors of G3BP-driven stress granule formation.

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the paralogs G3BP1 and G3BP2. G3BP1/2 proteins bind mRNAs and thereby promote the condensation of mRNPs into stress granules. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, referred to as G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is known to be targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress, and dissolve pre-existing stress granules when added to cells after stress granule formation. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent ideal tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

USB1 is a miRNA deadenylase that regulates hematopoietic development.

Mutations in the 3' to 5' RNA exonuclease USB1 cause hematopoietic failure in poikiloderma with neutropenia (PN). Although USB1 is known to regulate U6 small nuclear RNA maturation, the molecular mechanism underlying PN remains undetermined, as pre-mRNA splicing is unaffected in patients. We generated human embryonic stem cells harboring the PN-associated mutation c.531_delA in USB1 and show that this mutation impairs human hematopoiesis. Dysregulated microRNA (miRNA) levels in USB1 mutants during blood development contribute to hematopoietic failure, because of a failure to remove 3'-end adenylated tails added by PAPD5/7. Modulation of miRNA 3'-end adenylation through genetic or chemical inhibition of PAPD5/7 rescues hematopoiesis in USB1 mutants. This work shows that USB1 acts as a miRNA deadenylase and suggests PAPD5/7 inhibition as a potential therapy for PN.

Identification of PARN nuclease activity inhibitors by computational-based docking and high-throughput screening.

Poly(A)-specific ribonuclease (PARN) is a 3'-exoribonuclease that removes poly(A) tails from the 3' end of RNAs. PARN is known to deadenylate some ncRNAs, including hTR, Y RNAs, and some miRNAs and thereby enhance their stability by limiting the access of 3' to 5' exonucleases recruited by oligo(A) tails. Several PARN-regulated miRNAs target p53 mRNA, and PARN knockdown leads to an increase of p53 protein levels in human cells. Thus, PARN inhibitors might be used to induce p53 levels in some human tumors and act as a therapeutic strategy to treat cancers caused by repressed p53 protein. Herein, we used computational-based molecular docking and high-throughput screening (HTS) to identify small molecule inhibitors of PARN. Validation with in vitro and cell-based assays, identified 4 compounds, including 3 novel compounds and pyrimidopyrimidin-2-one GNF-7, previously shown to be a Bcr-Abl inhibitor, as PARN inhibitors. These inhibitors can be used as tool compounds and as lead compounds for the development of improved PARN inhibitors.

Cytosolic condensates rich in polyserine define subcellular sites of tau aggregation.

Tau aggregates are a hallmark of multiple neurodegenerative diseases and can contain RNAs and RNA-binding proteins, including serine/arginine repetitive matrix protein 2 (SRRM2) and pinin (PNN). However, how these nuclear proteins mislocalize and their influence on the prion-like propagation of tau aggregates is unknown. We demonstrate that polyserine repeats in SRRM2 and PNN are necessary and sufficient for recruitment to tau aggregates. Moreover, we show tau aggregates preferentially grow in association with endogenous cytoplasmic assemblies-mitotic interchromatin granules and cytoplasmic speckles (CSs)-which contain SRRM2 and PNN. Polyserine overexpression in cells nucleates assemblies that are sites of tau aggregate growth. Further, modulating the levels of polyserine-containing proteins results in a corresponding change in tau aggregation. These findings define a specific protein motif, and cellular condensates, that promote tau aggregate propagation. As CSs form in induced pluripotent stem cell (iPSC) derived neurons under inflammatory or hyperosmolar stress, they may affect tau aggregate propagation in neurodegenerative disease.

ADAR1 limits stress granule formation through both translation-dependent and translation-independent mechanisms.

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.

Modeling the effectiveness of olfactory testing to limit SARS-CoV-2 transmission.

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.

Just 2% of SARS-CoV-2-positive individuals carry 90% of the virus circulating in communities.

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.

RNA partitioning into stress granules is based on the summation of multiple interactions.

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.

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.

Chemical inhibition of PAPD5/7 rescues telomerase function and hematopoiesis in dyskeratosis congenita.

Dyskeratosis congenita (DC) is a pediatric bone marrow failure syndrome caused by germline mutations in telomere biology genes. Mutations in DKC1 (the most commonly mutated gene in DC), the 3' region of TERC, and poly(A)-specific ribonuclease (PARN) cause reduced levels of the telomerase RNA component (TERC) by reducing its stability and accelerating TERC degradation. We have previously shown that depleting wild-type DKC1 levels by RNA interference or expression of the disease-associated A353V mutation in the DKC1 gene leads to decay of TERC, modulated by 3'-end oligoadenylation by noncanonical poly(A) polymerase 5 (PAPD5) followed by 3' to 5' degradation by EXOSC10. Furthermore, the constitutive genetic silencing of PAPD5 is sufficient to rescue TERC levels, restore telomerase function, and elongate telomeres in DKC1_A353V mutant human embryonic stem cells (hESCs). Here, we tested a novel PAPD5/7 inhibitor (RG7834), which was originally discovered in screens against hepatitis B viral loads in hepatic cells. We found that treatment with RG7834 rescues TERC levels, restores correct telomerase localization in DKC1 and PARN-depleted cells, and is sufficient to elongate telomeres in DKC1_A353V hESCs. Finally, treatment with RG7834 significantly improved definitive hematopoietic potential from DKC1_A353V hESCs, indicating that the chemical inhibition of PAPD5 is a potential therapy for patients with DC and reduced TERC levels.

Coupling of translation quality control and mRNA targeting 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.

Modulation of RNA Condensation by the DEAD-Box Protein eIF4A.

Stress granules are condensates of non-translating mRNAs and proteins involved in the stress response and neurodegenerative diseases. Stress granules form in part through intermolecular RNA-RNA interactions, and to better understand how RNA-based condensation occurs, we demonstrate that RNA is effectively recruited to the surfaces of RNA or RNP condensates in vitro. We demonstrate that, through ATP-dependent RNA binding, the DEAD-box protein eIF4A reduces RNA condensation in vitro and limits stress granule formation in cells. This defines a function for eIF4A to limit intermolecular RNA-RNA interactions in cells. These results establish an important role for eIF4A, and potentially other DEAD-box proteins, as ATP-dependent RNA chaperones that limit the condensation of RNA, analogous to the function of proteins like HSP70 in combatting protein aggregates.

Transcriptome-Wide Comparison of Stress Granules and P-Bodies Reveals that Translation Plays a Major Role in RNA Partitioning.

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.

Quantitative proteomics identifies proteins that resist translational repression and become dysregulated in ALS-FUS.

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.

15-Deoxy-Δ12,14-prostaglandin J2 promotes phosphorylation of eukaryotic initiation factor 2α and activates the integrated stress response.

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.