-
Nucleic Acids Research Sep 2023Two prominent cytoplasmic RNA granules, ubiquitous RNA-processing bodies (PB) and inducible stress granules (SG), regulate mRNA translation and are intimately related....
Two prominent cytoplasmic RNA granules, ubiquitous RNA-processing bodies (PB) and inducible stress granules (SG), regulate mRNA translation and are intimately related. In this study, we found that arsenite (ARS)-induced SG formed in a stepwise process is topologically and mechanically linked to PB. Two essential PB components, GW182 and DDX6, are repurposed under stress to play direct but distinguishable roles in SG biogenesis. By providing scaffolding activities, GW182 promotes the aggregation of SG components to form SG bodies. DEAD-box helicase DDX6 is also essential for the proper assembly and separation of PB from SG. DDX6 deficiency results in the formation of irregularly shaped 'hybrid' PB/SG granules with accumulated components of both PB and SG. Wild-type DDX6, but not its helicase mutant E247A, can rescue the separation of PB from SG in DDX6KO cells, indicating a requirement of DDX6 helicase activity for this process. DDX6 activity in biogenesis of both PB and SG in the cells under stress is further modulated by its interaction with two protein partners, CNOT1 and 4E-T, of which knockdown affects the formation of both PB and also SG. Together, these data highlight a new functional paradigm between PB and SG biogenesis during the stress.
Topics: Cytoplasmic Granules; DEAD-box RNA Helicases; Processing Bodies; RNA; RNA Processing, Post-Transcriptional; Stress Granules; Humans; Cell Line
PubMed: 37427791
DOI: 10.1093/nar/gkad585 -
PloS One 2023Messenger RNA processing bodies (P-bodies) are cytoplasmic membrane-free organelles that contain proteins involved in mRNA silencing, storage and decay. The mechanism by...
Messenger RNA processing bodies (P-bodies) are cytoplasmic membrane-free organelles that contain proteins involved in mRNA silencing, storage and decay. The mechanism by which P-body components interact and the factors that regulate the stability of these structures are incompletely understood. In this study, we used a fluorescence-based, two-hybrid assay to investigate interactions between P-body components that occur inside the cell. LSm14a, PATL1, XRN1, and NBDY were found to interact with the N-terminal, WD40-domain-containing portion of EDC4. The N-terminus of full-length PATL1 was required to mediate the interaction between EDC4 and DDX6. The C-terminal, alpha helix-domain- containing portion of EDC4 was sufficient to mediate interaction with DCP1a and CCHCR1. In the absence of endogenous P-bodies, caused by depletion of LSm14a or DDX6, expression of the portion of EDC4 that lacked the N-terminus retained the ability to form cytoplasmic dots that were indistinguishable from P-bodies at the level of UV light microscopy. Despite the absence of endogenous P-bodies, this portion of EDC4 was able to recruit DCP1a, CCHCR1 and EDC3 to cytoplasmic dots. The results of this study permit the development of a new model of P-body formation and suggest that the N-terminus of EDC4 regulates the stability of these structures.
Topics: Animals; Processing Bodies; Cell Membrane; Cytoplasm; Cytosol; Mammals; RNA, Messenger
PubMed: 36877681
DOI: 10.1371/journal.pone.0282496 -
Journal of the American Chemical Society May 2023Stress granules (SGs) and processing-bodies (PBs, P-bodies) are ubiquitous and widely studied ribonucleoprotein (RNP) granules involved in cellular stress response,...
Stress granules (SGs) and processing-bodies (PBs, P-bodies) are ubiquitous and widely studied ribonucleoprotein (RNP) granules involved in cellular stress response, viral infection, and the tumor microenvironment. While proteomic and transcriptomic investigations of SGs and PBs have provided insights into molecular composition, chemical tools to probe and modulate RNP granules remain lacking. Herein, we combine an immunofluorescence (IF)-based phenotypic screen with chemoproteomics to identify sulfonyl-triazoles (SuTEx) capable of preventing or inducing SG and PB formation through liganding of tyrosine (Tyr) and lysine (Lys) sites in stressed cells. Liganded sites were enriched for RNA-binding and protein-protein interaction (PPI) domains, including several sites found in RNP granule-forming proteins. Among these, we functionally validate G3BP1 Y40, located in the NTF2 dimerization domain, as a ligandable site that can disrupt arsenite-induced SG formation in cells. In summary, we present a chemical strategy for the systematic discovery of condensate-modulating covalent small molecules.
Topics: DNA Helicases; Poly-ADP-Ribose Binding Proteins; Cytoplasmic Granules; RNA Recognition Motif Proteins; Proteomics; RNA Helicases
PubMed: 37159397
DOI: 10.1021/jacs.3c00165 -
Seminars in Cell & Developmental Biology Mar 2024Stress Granules (SGs) and Processing-bodies (P-bodies) are biomolecular condensates formed in the cell with the highly conserved purpose of maintaining balance between... (Review)
Review
Stress Granules (SGs) and Processing-bodies (P-bodies) are biomolecular condensates formed in the cell with the highly conserved purpose of maintaining balance between storage, translation, and degradation of mRNA. This balance is particularly important when cells are exposed to different environmental conditions and adjustments have to be made in order for plants to respond to and tolerate stressful conditions. While P-bodies are constitutively present in the cell, SG formation is a stress-induced event. Typically thought of as protein-RNA aggregates, SGs and P-bodies are formed by a process called liquid-liquid phase separation (LLPS), and both their function and composition are very dynamic. Both foci are known to contain proteins involved in translation, protein folding, and ATPase activity, alluding to their roles in regulating mRNA and protein expression levels. From an RNA perspective, SGs and P-bodies primarily consist of mRNAs, though long non-coding RNAs (lncRNAs) have also been observed, and more focus is now being placed on the specific RNAs associated with these aggregates. Recently, metabolites such as nucleotides and amino acids have been reported in purified plant SGs with implications for the energetic dynamics of these condensates. Thus, even though the field of plant SGs and P-bodies is relatively nascent, significant progress has been made in understanding their composition and biological role in stress responses. In this review, we discuss the most recent discoveries centered around SG and P-body function and composition in plants.
Topics: Processing Bodies; Stress Granules; RNA, Messenger; Cytoplasmic Granules; Stress, Physiological
PubMed: 36464613
DOI: 10.1016/j.semcdb.2022.11.008 -
BioRxiv : the Preprint Server For... Jul 2023The intracellular environment is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cell physiology. When exposed to stress,...
The intracellular environment is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cell physiology. When exposed to stress, mRNAs released after translational arrest condense with RNA binding proteins, resulting in the formation of membraneless RNA protein (RNP) condensates known as processing bodies (P-bodies) and stress granules (SGs). However, the impact of the assembly of these condensates on the biophysical properties of the crowded cytoplasmic environment remains unclear. Here, we find that upon exposure to stress, polysome collapse and condensation of mRNAs increases mesoscale particle diffusivity in the cytoplasm. Increased mesoscale diffusivity is required for the efficient formation of Q-bodies, membraneless organelles that coordinate degradation of misfolded peptides that accumulate during stress. Additionally, we demonstrate that polysome collapse and stress granule formation has a similar effect in mammalian cells, fluidizing the cytoplasm at the mesoscale. We find that synthetic, light-induced RNA condensation is sufficient to fluidize the cytoplasm, demonstrating a causal effect of RNA condensation. Together, our work reveals a new functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to effectively respond to stressful conditions.
PubMed: 37398029
DOI: 10.1101/2023.05.30.542963 -
World Journal of Gastrointestinal... Feb 2019Trans-acting factors controlling mRNA fate are critical for the post-transcriptional regulation of inflammation-related genes, as well as for oncogene and tumor... (Review)
Review
Trans-acting factors controlling mRNA fate are critical for the post-transcriptional regulation of inflammation-related genes, as well as for oncogene and tumor suppressor expression in human cancers. Among them, a group of RNA-binding proteins called "Adenylate-Uridylate-rich elements binding proteins" (AUBPs) control mRNA stability or translation through their binding to AU-rich elements enriched in the 3'UTRs of inflammation- and cancer-associated mRNA transcripts. AUBPs play a central role in the recruitment of target mRNAs into small cytoplasmic foci called Processing-bodies and stress granules (also known as P-body/SG). Alterations in the expression and activities of AUBPs and P-body/SG assembly have been observed to occur with colorectal cancer (CRC) progression, indicating the significant role AUBP-dependent post-transcriptional regulation plays in controlling gene expression during CRC tumorigenesis. Accordingly, these alterations contribute to the pathological expression of many early-response genes involved in prostaglandin biosynthesis and inflammation, along with key oncogenic pathways. In this review, we summarize the current role of these proteins in CRC development. CRC remains a major cause of cancer mortality worldwide and, therefore, targeting these AUBPs to restore efficient post-transcriptional regulation of gene expression may represent an appealing therapeutic strategy.
PubMed: 30788036
DOI: 10.4251/wjgo.v11.i2.71 -
PLoS Pathogens Aug 2022A dysregulated proinflammatory cytokine response is characteristic of severe coronavirus infections caused by SARS-CoV-2, yet our understanding of the underlying...
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.
Topics: COVID-19; Coronavirus OC43, Human; Cytokines; Humans; In Situ Hybridization, Fluorescence; Processing Bodies; RNA; SARS-CoV-2
PubMed: 35998203
DOI: 10.1371/journal.ppat.1010724 -
RNA Biology Jun 2017Aside from nucleoli, Cajal bodies (CBs) are the best-characterized organelles of mammalian cell nuclei. Like nucleoli, CBs concentrate ribonucleoproteins (RNPs), in... (Review)
Review
Aside from nucleoli, Cajal bodies (CBs) are the best-characterized organelles of mammalian cell nuclei. Like nucleoli, CBs concentrate ribonucleoproteins (RNPs), in particular, spliceosomal small nuclear RNPs (snRNPs) and small nucleolar RNPs (snoRNPs). In one of the best-defined functions of CBs, most of the snoRNPs are involved in site-specific modification of snRNAs. The two major modifications are pseudouridylation and 2'-O-methylation that are guided by the box H/ACA and C/D snoRNPs, respectively. This review details the modifications, their function, the mechanism of modification, and the machineries involved. We dissect the different classes of noncoding RNAs that meet in CBs, guides and substrates. Open questions and conundrums, often raised and appearing due to experimental limitations, are pointed out and discussed. The emphasis of the review is on mammalian CBs and their function in modification of noncoding RNAs.
Topics: Animals; Coiled Bodies; Humans; RNA Processing, Post-Transcriptional; RNA, Small Nuclear; RNA, Small Nucleolar; Ribonucleoproteins, Small Nucleolar; Spliceosomes
PubMed: 27775477
DOI: 10.1080/15476286.2016.1249091 -
PLoS Biology Jul 2023Microglia-mediated neuroinflammation is involved in various neurological diseases, including ischemic stroke, but the endogenous mechanisms preventing unstrained...
Microglia-mediated neuroinflammation is involved in various neurological diseases, including ischemic stroke, but the endogenous mechanisms preventing unstrained inflammation is still unclear. The anti-inflammatory role of transcription factor nuclear receptor subfamily 4 group A member 1 (NR4A1) in macrophages and microglia has previously been identified. However, the endogenous mechanisms that how NR4A1 restricts unstrained inflammation remain elusive. Here, we observed that NR4A1 is up-regulated in the cytoplasm of activated microglia and localizes to processing bodies (P-bodies). In addition, we found that cytoplasmic NR4A1 functions as an RNA-binding protein (RBP) that directly binds and destabilizes Tnf mRNA in an N6-methyladenosine (m6A)-dependent manner. Remarkably, conditional microglial deletion of Nr4a1 elevates Tnf expression and worsens outcomes in a mouse model of ischemic stroke, in which case NR4A1 expression is significantly induced in the cytoplasm of microglia. Thus, our study illustrates a novel mechanism that NR4A1 posttranscriptionally regulates Tnf expression in microglia and determines stroke outcomes.
Topics: Animals; Mice; Transcription Factors; Microglia; Stroke; Ischemic Stroke; Inflammation; RNA, Messenger
PubMed: 37486903
DOI: 10.1371/journal.pbio.3002199 -
Neuroscience and Biobehavioral Reviews Feb 2021The medial diencephalon, in particular the mammillary bodies and anterior thalamic nuclei, has long been linked to memory and amnesia. The mammillary bodies provide a... (Review)
Review
The medial diencephalon, in particular the mammillary bodies and anterior thalamic nuclei, has long been linked to memory and amnesia. The mammillary bodies provide a dense input into the anterior thalamic nuclei, via the mammillothalamic tract. In both animal models, and in patients, lesions of the mammillary bodies, mammillothalamic tract and anterior thalamic nuclei all produce severe impairments in temporal and contextual memory, yet it is uncertain why these regions are critical. Mounting evidence from electrophysiological and neural imaging studies suggests that mammillothalamic projections exercise considerable distal influence over thalamo-cortical and hippocampo-cortical interactions. Here, we outline how damage to the mammillary body-anterior thalamic axis, in both patients and animal models, disrupts behavioural performance on tasks that relate to contextual ("where") and temporal ("when") processing. Focusing on the medial mammillary nuclei as a possible 'theta-generator' (through their interconnections with the ventral tegmental nucleus of Gudden) we discuss how the mammillary body-anterior thalamic pathway may contribute to the mechanisms via which the hippocampus and neocortex encode representations of experience.
Topics: Amnesia; Animals; Anterior Thalamic Nuclei; Humans; Mammillary Bodies; Memory; Neural Pathways
PubMed: 33309908
DOI: 10.1016/j.neubiorev.2020.11.031