Cytoplasmic Ribonucleoprotein Research Articles

Major Vault Protein/Lung Resistance-Related Protein: A Novel Biomarker for Inflammation and Acute Infections.

Vault particles are large cytoplasmic ribonucleoprotein particles that participate in inflammation. The aim of this study was to assess the diagnostic and prognostic value of major vault protein (MVP) in patients with inflammation, in order to determine whether MVP could be used as a biomarker for infection or inflammation. We also aimed to compare the diagnostic impact of MVP compared to other conventional measurements, such as CRP or white blood cell (WBC) counts. CRP and MVP levels were measured in 111 sera samples from 85 patients with inflammation admitted to a tertiary-care hospital and 26 healthy individuals during an 18-month period (2019-2020), using nephelometry and a custom MVP sandwich ELISA assay, respectively. In addition, WBC counts were measured using a commercial assay. MVP levels were found to be elevated in patients with inflammation compared to healthy individuals (p < 0.0001). Moreover, MVP levels were higher in patients with inflammation due to an infectious etiology compared to those with non-infectious etiology (p = 0.0006). MVP levels significantly decreased during the first four days of infection in response to antibiotic treatment, while CRP levels showed a less-sensitive decline. An ROC curve analysis demonstrated that MVP and CRP have similarly high diagnostic accuracy, with AUCs of 0.955 and 0.995, respectively, followed by WBCs with an AUC of 0.805. The ROC curves demonstrated that MVP has the potential to serve as a diagnostic biomarker for inflammation and infection. Additionally, MVP levels may reflect the efficacy of antibiotic treatment.

SRP54 of black carp negatively regulates MDA5-mediated antiviral innate immunity

Signal Recognition Particle 54 kDa (SRP54) is a subunit of the signal recognition particle (SRP), a cytoplasmic ribonucleoprotein complex guiding the transportation of newly synthesized proteins from polyribosomes to endoplasmic reticulum. In mammals, it has been reported to regulate the RLR signaling pathway negatively by impairing the association between MAVS and MDA5/RIG-I. However, the role of SRP54 in teleost antiviral innate immune response remains obscure. In this study, the SRP54 homolog of black carp (bcSRP54) has been cloned, and its function in antiviral innate immunity has been elucidated. The CDS of bcSRP54 gene consists of 1515 nucleotides and encodes 504 amino acids. Immunofluorescence (IF) showed that bcSRP54 was mainly distributed in the cytoplasm. Overexpressed bcSRP54 significantly reduced bcMDA5-mediated transcription of interferon (IFN) promoter in reporter assay. Co-expression of bcSRP54 and bcMDA5 significantly suppressed bcMDA5-mediated IFN signaling and antiviral activity, while bcSRP54 knockdown increased the antiviral ability of host cells. In addition, the results of the immunofluorescence staining demonstrated the subcellular overlapping between bcSRP54 and bcMDA5, and the co-immunoprecipitation (co-IP) experiment identified their association. Furthermore, the over-expression of bcSRP54 did not influence the protein expression and ubiquitination modification level of bcMDA5, however, hindered the binding of bcMDA5 to bcMAVS. In summary, our results conclude that bcSRP54 targets bcMDA5 and inhibits the interaction between bcMDA5 and bcMAVS, thereby negatively regulating antiviral innate immunity, which provides insight into how teleost SRP54 regulates IFN signaling.

Nanoscale dynamics and localization of single endogenous mRNAs in stress granules.

Stress granules (SGs) are cytoplasmic messenger ribonucleoprotein granules transiently formed in stressed mammalian cells. Although SG components have been well characterized, detailed insights into the molecular behavior inside SGs remain unresolved. We investigated nanoscale dynamics and localization of endogenous mRNAs in SGs combining single mRNA tracking and super-resolution localization microscopy. First, we developed a methodology for tracking single mRNAs within SGs, revealing that although mRNAs in SGs are mainly stationary (∼40%), they also move in a confined (∼25%) or freely diffusing (∼35%) manner. Second, the super-resolution localization microscopy showed that the mRNAs in SGs are heterogeneously distributed and partially form high-density clusters. Third, we simultaneously performed single mRNA tracking and super-resolution microscopy in SGs, demonstrating that single mRNA trajectories are mainly found around high-density clusters. Finally, a quantitative analysis of mRNA localization and dynamics during stress removal was conducted using live super-resolution imaging and single-molecule tracking. These results suggest that SGs have a highly organized structure that enables dynamic regulation of the mRNAs at the nanoscale, which is responsible for the ordered formation and the wide variety of functions of SGs.

Impact of protein domains on the MEL2 granule, a cytoplasmic ribonucleoprotein complex maintaining faithful meiosis progression in rice.

Cytoplasmic ribonucleoprotein (RNP) granules are membraneless structures composed of various RNAs and proteins that play important roles in post-transcriptional regulation. While RNP granules are known to regulate the meiotic entry in some organisms, little is known about their roles in plants. In this study, we observed the cytoplasmic granular structures of rice RNA-binding protein MEIOSIS ARRESTED AT LEPTOTENE2 (MEL2), which contributes to the control of meiotic entry timing, in leaf protoplasts and spore mother cells. We performed colocalization analysis with known cytoplasmic RNP factors, and domain deletion analysis to assess their impact on granule formation and meiosis progression. Conservation of MEL2 domains across plant species was also explored. Our results indicated that MEL2 granules colocalized with processing body and stress granule factors. The maintenance of granule properties modulated by LOTUS domain and the intrinsically disordered region (IDR) is essential for proper MEL2 function in meiosis progression. MEL2-like proteins widely found in plant kingdom conserved LOTUS domain followed by the IDR despite their diverse domain structures, suggesting the functional conservation of these domains among plant species. This study highlights the role of MEL2 granule dynamics and its impact on meiotic transition and progression.

Abstract 7480: Human LINE-1 ORF1p retrotransposition independent function in pancreatic cancer

Abstract The aberrant expression of repeat elements is a common feature found in both mouse models and human pancreatic ductal adenocarcinoma (PDAC). The majority of these are non-coding RNAs that have shown the ability to stimulate a viral-like innate immune response. The long interspersed nuclear element 1 (LINE-1) retrotransposon is one of the few protein coding repeat elements in the human genome. LINE-1 has two open reading frames, ORF1 and ORF2. The former (ORF1p) encodes a ribonucleotide protein and the latter (ORF2p) a reverse transcriptase and endonuclease. Although nuclear ORF1p and ORF2p is required for retrotransposition, ORF1p is primarily localized in the cytoplasm, which suggests the possibility of ORF1p function independent of LINE-1 retrotransposition. Here, we show that ORF1p has a function to shield repeat RNAs from activating pathogen recognition receptor (PRR) mediated antiviral response in PDAC cells. LINE-1 ORF1p-targeting shRNA significantly inhibits patient-derived PDAC tumor growth both in vitro and in vivo. Total RNA sequencing (RNA-seq) reveals distinct transcriptional profiles with ORF1p deficiency. Gene set enrichment analysis (GSEA) identified high enrichment of innate immune response-related pathways including IL6-JAK-STAT3 signaling, inflammatory response, interferon response, and apoptosis in ORF1 knockdown PDAC cell lines. These immune related pathways are suppressed by deletion of RIG-I and MAVS, two components of viral RNA sensing. Mechanistically, we demonstrated that ORF1p is associated with repeat RNAs including LINE-1 RNA (L1HS) in processing bodies (PBs), which are cytoplasmic membraneless ribonucleoprotein (RNP) granules. In the PBs, ORF1p interacts with MOV10 RNA helicase, an essential enzyme for dsRNA unwinding. Using MOV10 truncated mutants, we found that the ORF1p-MOV10 interaction is important in attenuation of PRR sensing and apoptosis upon repeat RNA exposure. Finally, we evalulated the activation of the innate immune response in the tumor microenvironment by ORF1p depletion. These results suggest that ORF1p loss induces a desmoplastic reaction driven by Acta2+ myofibroblastic CAFs in response to inflammatory cytokines. Collectively, our results demonstrate a critical and previously uncharacterized role of ORF1p in evasion of endogenous repeat RNA-triggered antiviral immune response in PDAC cells with implications in the tumor microenvironment. Citation Format: Eunae You, Bidish Patel, Alexandra Rojas, Natalie Ho, Siyu Sun, Michael Raabe, Yuhui Song, Ildiko Phillips, Katherine Xu, Peter Richieri, Linda Nieman, Benjamin Greenbaum, David Ting. Human LINE-1 ORF1p retrotransposition independent function in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7480.

Defining Distinct RNA-Protein Interactomes of SARS-CoV-2 Genomic and Subgenomic RNAs.

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. Individual interactomes indicated viral associations with cell response pathways, including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We tested the significance of three protein interactors in these pathways (APOBEC3F, PPP1CC, and MSI2) using siRNA knockdowns, with several knockdowns affecting viral gene expression, most consistently PPP1CC. This study describes a new technology for high-resolution studies of SARS-CoV-2 RNA regulation and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.

Dysregulation of alternative splicing underlies synaptic defects in familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by the degeneration of upper and lower motor neurons, progressive wasting and paralysis of voluntary muscles. A hallmark of ALS is the frequent nuclear loss and cytoplasmic accumulation of RNA binding proteins (RBPs) in motor neurons (MN), which leads to aberrant alternative splicing regulation. However, whether altered splicing patterns are also present in familial models of ALS without mutations in RBP-encoding genes has not been investigated yet. Herein, we found that altered splicing of synaptic genes is a common trait of familial ALS MNs. Similar deregulation was also observed in hSOD1G93A MN-like cells. In silico analysis identified the potential regulators of these pre-mRNAs, including the RBP Sam68. Immunofluorescence analysis and biochemical fractionation experiments revealed that Sam68 accumulates in the cytoplasmic insoluble ribonucleoprotein fraction of MN. Remarkably, the synaptic splicing events deregulated in ALS MNs were also affected in Sam68-/- spinal cords. Recombinant expression of Sam68 protein was sufficient to rescue these splicing changes in ALS hSOD1G93A MN-like cells. Hence, our study highlights an aberrant function of Sam68, which leads to splicing changes in synaptic genes and may contribute to the MN phenotype that characterizes ALS.

Stress granule-mediated RNA regulatory mechanism in Alzheimer's disease.

Living organisms experience a range of stresses. To cope effectively with these stresses, eukaryotic cells have evolved a sophisticated mechanism involving the formation of stress granules (SGs), which play a crucial role in protecting various types of RNA species under stress, such as mRNAs and long non-coding RNAs (lncRNAs). SGs are non-membranous cytoplasmic ribonucleoprotein (RNP) granules, and the RNAs they contain are translationally stalled. Importantly, SGs have been thought to contribute to the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD). SGs also contain multiple RNA-binding proteins (RBPs), several of which have been implicated in AD progression. SGs are transient structures that dissipate after stress relief. However, the chronic stresses associated with aging lead to the persistent formation of SGs and subsequently to solid-like pathological SGs, which could impair cellular RNA metabolism and also act as a nidus for the aberrant aggregation of AD-associated proteins. In this paper, we provide a comprehensive summary of the physical basis of SG-enriched RNAs and SG-resident RBPs. We then review the characteristics of AD-associated gene transcripts and their similarity to the SG-enriched RNAs. Furthermore, we summarize and discuss the functional implications of SGs in neuronal RNA metabolism and the aberrant aggregation of AD-associated proteins mediated by SG-resident RBPs in the context of AD pathogenesis. Geriatr Gerontol Int 2024; 24: 7-14.

Phase separation and pathologic transitions of RNP condensates in neurons: implications for amyotrophic lateral sclerosis, frontotemporal dementia and other neurodegenerative disorders.

Liquid-liquid phase separation results in the formation of dynamic biomolecular condensates, also known as membrane-less organelles, that allow for the assembly of functional compartments and higher order structures within cells. Multivalent, reversible interactions between RNA-binding proteins (RBPs), including FUS, TDP-43, and hnRNPA1, and/or RNA (e.g., RBP-RBP, RBP-RNA, RNA-RNA), result in the formation of ribonucleoprotein (RNP) condensates, which are critical for RNA processing, mRNA transport, stability, stress granule assembly, and translation. Stress granules, neuronal transport granules, and processing bodies are examples of cytoplasmic RNP condensates, while the nucleolus and Cajal bodies are representative nuclear RNP condensates. In neurons, RNP condensates promote long-range mRNA transport and local translation in the dendrites and axon, and are essential for spatiotemporal regulation of gene expression, axonal integrity and synaptic function. Mutations of RBPs and/or pathologic mislocalization and aggregation of RBPs are hallmarks of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease. ALS/FTD-linked mutations of RBPs alter the strength and reversibility of multivalent interactions with other RBPs and RNAs, resulting in aberrant phase transitions. These aberrant RNP condensates have detrimental functional consequences on mRNA stability, localization, and translation, and ultimately lead to compromised axonal integrity and synaptic function in disease. Pathogenic protein aggregation is dependent on various factors, and aberrant dynamically arrested RNP condensates may serve as an initial nucleation step for pathologic aggregate formation. Recent studies have focused on identifying mechanisms by which neurons resolve phase transitioned condensates to prevent the formation of pathogenic inclusions/aggregates. The present review focuses on the phase separation of neurodegenerative disease-linked RBPs, physiological functions of RNP condensates, and the pathologic role of aberrant phase transitions in neurodegenerative disease, particularly ALS/FTD. We also examine cellular mechanisms that contribute to the resolution of aberrant condensates in neurons, and potential therapeutic approaches to resolve aberrantly phase transitioned condensates at a molecular level.

Syndecan-4 is the key proteoglycan involved in mediating sepsis-associated lung injury

Vascular endothelial cell dysfunction involving syndecan (SDC) proteoglycans contributes to acute sepsis-associated lung injury (ALI), but the exact SDC isoform involved is unclear. We aimed to clarify which SDCs are involved in ALI. A relevant gene expression dataset (GSE5883) was analysed for differentially expressed genes (DEGs) between lipopolysaccharide (LPS)-treated and control lung endothelial cells and for SDC isoform expression. Bioinformatic analyses to predict DEG function were conducted using R language, Gene Ontology, and the Kyoto Encyclopedia of Genes and Genomes. SDC2 and SDC4 expression profiles were examined under inflammatory conditions in human lung vascular endothelial cell and mouse sepsis-associated ALI models. Transcription factors regulating SDC2/4 were predicted to indirectly assess SDC involvement in septic inflammation. Of the DEGs, 224 and 102 genes were up- and downregulated, respectively. Functional analysis indicated that DEGs were involved in modulating receptor ligand and signalling receptor activator activities, cytokine receptor binding, responses to LPS and molecules of bacterial origin, regulation of cell adhesion, tumour necrosis factor signalling, and other functions. DEGs were also enriched for cytoplasmic ribonucleoprotein granules, transcription regulator complexes, and membrane raft cellular components. SDC4 gene expression was 4.5-fold higher in the LPS group than in the control group, while SDC2 levels were similar in both groups. SDC4 mRNA and protein expression was markedly upregulated in response to inflammatory injury, and SDC4 downregulation severely exacerbated inflammatory responses in both in vivo and in vitro models. Overall, our data demonstrate that SDC4, rather than SDC2, is involved in LPS-induced sepsis-associated ALI.

Regulation of Miwi-mediated mRNA stabilization by Ck137956/Tssa is essential for male fertility

BackgroundSperm is formed through spermiogenesis, a highly complex process involving chromatin condensation that results in cessation of transcription. mRNAs required for spermiogenesis are transcribed at earlier stages and translated in a delayed fashion during spermatid formation. However, it remains unknown that how these repressed mRNAs are stabilized.ResultsHere we report a Miwi-interacting testis-specific and spermiogenic arrest protein, Ck137956, which we rename Tssa. Deletion of Tssa led to male sterility and absence of sperm formation. The spermiogenesis arrested at the round spermatid stage and numerous spermiogenic mRNAs were down-regulated in Tssa−/− mice. Deletion of Tssa disrupted the localization of Miwi to chromatoid body, a specialized assembly of cytoplasmic messenger ribonucleoproteins (mRNPs) foci present in germ cells. We found that Tssa interacted with Miwi in repressed mRNPs and stabilized Miwi-interacting spermiogenesis-essential mRNAs.ConclusionsOur findings indicate that Tssa is indispensable in male fertility and has critical roles in post-transcriptional regulations by interacting with Miwi during spermiogenesis.

FAM69C functions as a kinase for eIF2α and promotes stress granule assembly

Stress granules are dynamic cytoplasmic ribonucleoprotein granules that assemble in response to cellular stress. Aberrant formation of stress granules has been linked to neurodegenerative diseases. However, the molecular mechanisms underlying the initiation of stress granules remain elusive. Here we report that the brain‐enriched protein kinase FAM69C promotes stress granule assembly through phosphorylation of eukaryotic translation initiation factor 2 (eIF2α). FAM69C physically interacts with eIF2α and functions as a stress‐specific kinase for eIF2α, leading to stress‐induced protein translation arrest and stress granule assembly. Primary microglia derived from Fam69c knockout mice exhibit aberrant stress granule assembly in response to oxidative stress and ATP. Defective stress granule assembly in microglia correlates with the formation of ASC specks and NLRP3 inflammasome activation, whereas induction of stress granule precludes inflammasome formation. Consistently, increased NLRP3 levels, caspase‐1 cleavage and Il18 expression corroborate microglia‐associated neuroinflammation in aged Fam69c knockout mice. Our study demonstrates that FAM69C is critical for stress granule assembly and suggests its role in the regulation of microglia function.

Kaposi's sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes the inflammatory and angiogenic endothelial cell neoplasm, Kaposi's sarcoma (KS). We previously demonstrated that the KSHV Kaposin B (KapB) protein promotes inflammation via the disassembly of cytoplasmic ribonucleoprotein granules called processing bodies (PBs). PBs modify gene expression by silencing or degrading labile messenger RNAs (mRNAs), including many transcripts that encode inflammatory or angiogenic proteins associated with KS disease. Although our work implicated PB disassembly as one of the causes of inflammation during KSHV infection, the precise mechanism used by KapB to elicit PB disassembly was unclear. Here we reveal a new connection between the degradative process of autophagy and PB disassembly. We show that both latent KSHV infection and KapB expression enhanced autophagic flux via phosphorylation of the autophagy regulatory protein, Beclin. KapB was necessary for this effect, as infection with a recombinant virus that does not express the KapB protein did not induce Beclin phosphorylation or autophagic flux. Moreover, we showed that PB disassembly mediated by KSHV or KapB, depended on autophagy genes and the selective autophagy receptor NDP52/CALCOCO2 and that the PB scaffolding protein, Pat1b, co-immunoprecipitated with NDP52. These studies reveal a new role for autophagy and the selective autophagy receptor NDP52 in promoting PB turnover and the concomitant synthesis of inflammatory molecules during KSHV infection.

Drosophila Me31B is a Dual eIF4E-Interacting Protein

Eukaryotic translation initiation factor 4E (eIF4E) is a key factor involved in different aspects of mRNA metabolism. Drosophila melanogaster genome encodes eight eIF4E isoforms, and the canonical isoform eIF4E-1 is a ubiquitous protein that plays a key role in mRNA translation. eIF4E-3 is specifically expressed in testis and controls translation during spermatogenesis. In eukaryotic cells, translational control and mRNA decay is highly regulated in different cytoplasmic ribonucleoprotein foci, which include the processing bodies (PBs). In this study, we show that Drosophila eIF4E-1 and eIF4E-3 occur in PBs along the DEAD-box RNA helicase Me31B. We show that Me31B interacts with eIF4E-1 and eIF4E-3 by means of yeast two-hybrid system, FRET in D. melanogaster S2 cells and coimmunoprecipitation in testis. Truncation and point mutations of Me31B proteins show two eIF4E-binding sites located in different protein domains. Residues Y401-L407 (at the carboxy-terminus) are essential for interaction with eIF4E-1, whereas residues F63-L70 (at the amino-terminus) are critical for interaction with eIF4E-3. The residue W117 in eIF4E-1 and the homolog position F103 in eIF4E-3 are necessary for Me31B-eIF4E interaction suggesting that the change of tryptophan to phenylalanine provides specificity. Me31B represents a novel type of eIF4E-interacting protein with dual and specific interaction domains that might be recognized by different eIF4E isoforms in different tissues, adding complexity to the control of gene expression in eukaryotes.

Intermitochondrial cement (IMC) harbors piRNA biogenesis machinery and exonuclease domain-containing proteins EXD1 and EXD2 in mouse spermatocytes.

Germ granules are large cytoplasmic ribonucleoprotein complexes that emerge in the germline to participate in RNA regulation. The two most prominent germ granules are the intermitochondrial cement (IMC) in meiotic spermatocytes and the chromatoid body (CB) in haploid round spermatids, both functionally linked to the PIWI-interacting RNA (piRNA) pathway. In this study, we clarified the IMC function by identifying proteins that form complexes with a well-known IMC protein PIWIL2/MILI in the mouse testis. The PIWIL2 interactome included several proteins with known functions in piRNA biogenesis. We further characterized the expression and localization of two of the identified proteins, Exonuclease 3'-5' domain-containing proteins EXD1 and EXD2, and confirmed their localization to the IMC. We showed that EXD2 interacts with PIWIL2, and that the mutation of Exd2 exonuclease domain in mice induces misregulation of piRNA levels originating from specific pachytene piRNA clusters, but does not disrupt male fertility. Altogether, this study highlights the central role of the IMC as a platform for piRNA biogenesis, and suggests that EXD1 and EXD2 function in the IMC-mediated RNA regulation in postnatal male germ cells.

Relocalization of Translation Termination and Ribosome Recycling Factors to Stress Granules Coincides with Elevated Stop-Codon Readthrough and Reinitiation Rates upon Oxidative Stress.

Upon oxidative stress, mammalian cells rapidly reprogram their translation. This is accompanied by the formation of stress granules (SGs), cytoplasmic ribonucleoprotein condensates containing untranslated mRNA molecules, RNA-binding proteins, 40S ribosomal subunits, and a set of translation initiation factors. Here we show that arsenite-induced stress causes a dramatic increase in the stop-codon readthrough rate and significantly elevates translation reinitiation levels on uORF-containing and bicistronic mRNAs. We also report the recruitment of translation termination factors eRF1 and eRF3, as well as ribosome recycling and translation reinitiation factors ABCE1, eIF2D, MCT-1, and DENR to SGs upon arsenite treatment. Localization of these factors to SGs may contribute to a rapid resumption of mRNA translation after stress relief and SG disassembly. It may also suggest the presence of post-termination, recycling, or reinitiation complexes in SGs. This new layer of translational control under stress conditions, relying on the altered spatial distribution of translation factors between cellular compartments, is discussed.

Cytoplasmic ribonucleoprotein complexes, RNA helicases and coronavirus infection

RNA metabolism in the eukaryotic cell includes the formation of ribonucleoprotein complexes (RNPs) that, depending on their protein components, have a different function. Cytoplasmic RNPs, such as stress granules (SGs) or P-bodies (PBs) are quite relevant during infections modulating viral and cellular RNA expression and as key players in the host cell antiviral response. RNA helicases are abundant components of RNPs and could have a significant effect on viral infection. This review focuses in the role that RNPs and RNA helicases have during coronavirus (CoVs) infection. CoVs are emerging highly pathogenic viruses with a large single-stranded RNA genome. During CoV infection, a complex network of RNA-protein interactions in different RNP structures is established. In general, RNA helicases and RNPs have an antiviral function, but there is limited knowledge on whether the viral protein interactions with cell components are mediators of this antiviral effect or are part of the CoV antiviral counteraction mechanism. Additional data is needed to elucidate the role of these RNA-protein interactions during CoV infection and their potential contribution to viral replication or pathogenesis.

A circadian clock translational control mechanism targets specific mRNAs to cytoplasmic messenger ribonucleoprotein granules

Phosphorylation of Neurospora crassa eukaryotic initiation factor 2 α (eIF2α), a conserved translation initiation factor, is clock controlled. To determine the impact of rhythmic eIF2α phosphorylation on translation, we performed temporal ribosome profiling and RNA sequencing (RNA-seq) in wild-type (WT), clock mutant Δfrq, eIF2α kinase mutant Δcpc-3, and constitutively active cpc-3c cells. About 14% of mRNAs are rhythmically translated in WT cells, and translation rhythms for ∼30% of these mRNAs, which we named circadian translation-initiation-controlled genes (cTICs), are dependent on the clock and CPC-3. Most cTICs are expressed from arrhythmic mRNAs and contain a P-body (PB) localization motif in their 5' leader sequence. Deletion of SNR-1, a component of cytoplasmic messenger ribonucleoprotein granules (cmRNPgs) that include PBs and stress granules (SGs), and the PB motif on one of the cTIC mRNAs, zip-1, significantly alters zip-1 rhythmic translation. These results reveal that the clock regulates rhythmic translation of specific mRNAs through rhythmic eIF2α activity and cmRNPg metabolism.

SpotitPy: a semi-automated tool for object-based co-localization of fluorescent labels in microscopy images

BackgroundIn fluorescence microscopy, co-localization refers to the spatial overlap between different fluorescent labels in cells. The degree of overlap between two or more channels in a microscope may reveal a physical interaction or topological functional interconnection between molecules. Recent advances in the imaging field require the development of specialized computational analysis software for the unbiased assessment of fluorescently labelled microscopy images.ResultsHere we present SpotitPy, a semi-automated image analysis tool for 2D object-based co-localization. SpotitPy allows the user to select fluorescent labels and perform a semi-automated and robust segmentation of the region of interest in distinct cell types. The workflow integrates advanced pre-processing manipulations for de-noising and in-depth semi-automated quantification of the co-localized fluorescent labels in two different channels. We validated SpotitPy by quantitatively assessing the presence of cytoplasmic ribonucleoprotein granules, e.g. processing (P) bodies, under conditions that challenge mRNA translation, thus highlighting SpotitPy benefits for semi-automatic, accurate analysis of large image datasets in eukaryotic cells. SpotitPy comes in a command line interface or a simple graphical user interphase and can be used as a standalone application.ConclusionsOverall, we present a novel and user-friendly tool that performs a semi-automated image analysis for 2D object-based co-localization. SpotitPy can provide reproducible and robust quantifications for large datasets within a limited timeframe. The software is open-source and can be found in the GitHub project repository: (https://github.com/alexiaales/SpotitPy).

Human coronaviruses disassemble processing bodies.

A dysregulated proinflammatory cytokine response is characteristic of severe coronavirus infections caused by SARS-CoV-2, yet our understanding of the underlying mechanism responsible for this imbalanced immune response remains incomplete. Processing bodies (PBs) are cytoplasmic membraneless ribonucleoprotein granules that control innate immune responses by mediating the constitutive decay or suppression of mRNA transcripts, including many that encode proinflammatory cytokines. PB formation promotes turnover or suppression of cytokine RNAs, whereas PB disassembly corresponds with the increased stability and/or translation of these cytokine RNAs. Many viruses cause PB disassembly, an event that can be viewed as a switch that rapidly relieves cytokine RNA repression and permits the infected cell to respond to viral infection. Prior to this submission, no information was known about how human coronaviruses (CoVs) impacted PBs. Here, we show SARS-CoV-2 and the common cold CoVs, OC43 and 229E, induced PB loss. We screened a SARS-CoV-2 gene library and identified that expression of the viral nucleocapsid (N) protein from SARS-CoV-2 was sufficient to mediate PB disassembly. RNA fluorescent in situ hybridization revealed that transcripts encoding TNF and IL-6 localized to PBs in control cells. PB loss correlated with the increased cytoplasmic localization of these transcripts in SARS-CoV-2 N protein-expressing cells. Ectopic expression of the N proteins from five other human coronaviruses (OC43, MERS, 229E, NL63 and SARS-CoV) did not cause significant PB disassembly, suggesting that this feature is unique to SARS-CoV-2 N protein. These data suggest that SARS-CoV-2-mediated PB disassembly contributes to the dysregulation of proinflammatory cytokine production observed during severe SARS-CoV-2 infection.