Competing Protein-RNA Interaction Networks Control Multiphase Intracellular Organization.

Liquid-liquid phase separation (LLPS) mediates formation of membraneless condensates such as those associated with RNA processing, but the rules that dictate their assembly, substructure, and coexistence with other liquid-like compartments remain elusive. Here, we address the biophysical mechanism of this multiphase organization using quantitative reconstitution of cytoplasmic stress granules (SGs) with attached P-bodies in human cells. Protein-interaction networks can be viewed as interconnected complexes (nodes) of RNA-binding domains (RBDs), whose integrated RNA-binding capacity determines whether LLPS occurs upon RNA influx. Surprisingly, both RBD-RNA specificity and disordered segments of key proteins are non-essential, but modulate multiphase condensation. Instead, stoichiometry-dependent competition between protein networks for connecting nodes determines SG and P-body composition and miscibility, while competitive binding of unconnected proteins disengages networks and prevents LLPS. Inspired by patchy colloid theory, we propose a general framework by which competing networks give rise to compositionally specific and tunable condensates, while relative linkage between nodes underlies multiphase organization.

Stress granules are shock absorbers that prevent excessive innate immune responses to dsRNA.

Proper defense against microbial infection depends on the controlled activation of the immune system. This is particularly important for the RIG-I-like receptors (RLRs), which recognize viral dsRNA and initiate antiviral innate immune responses with the potential of triggering systemic inflammation and immunopathology. Here, we show that stress granules (SGs), molecular condensates that form in response to various stresses including viral dsRNA, play key roles in the controlled activation of RLR signaling. Without the SG nucleators G3BP1/2 and UBAP2L, dsRNA triggers excessive inflammation and immune-mediated apoptosis. In addition to exogenous dsRNA, host-derived dsRNA generated in response to ADAR1 deficiency is also controlled by SG biology. Intriguingly, SGs can function beyond immune control by suppressing viral replication independently of the RLR pathway. These observations thus highlight the multi-functional nature of SGs as cellular "shock absorbers" that converge on protecting cell homeostasis by dampening both toxic immune response and viral replication.

Spatiotemporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis.

Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly.

Tumor Interferon Signaling Is Regulated by a lncRNA INCR1 Transcribed from the PD-L1 Locus.

Tumor interferon (IFN) signaling promotes PD-L1 expression to suppress T cell-mediated immunosurveillance. We identify the IFN-stimulated non-coding RNA 1 (INCR1) as a long noncoding RNA (lncRNA) transcribed from the PD-L1 locus and show that INCR1 controls IFNγ signaling in multiple tumor types. Silencing INCR1 decreases the expression of PD-L1, JAK2, and several other IFNγ-stimulated genes. INCR1 knockdown sensitizes tumor cells to cytotoxic T cell-mediated killing, improving CAR T cell therapy. We discover that PD-L1 and JAK2 transcripts are negatively regulated by binding to HNRNPH1, a nuclear ribonucleoprotein. The primary transcript of INCR1 binds HNRNPH1 to block its inhibitory effects on the neighboring genes PD-L1 and JAK2, enabling their expression. These findings introduce a mechanism of tumor IFNγ signaling regulation mediated by the lncRNA INCR1 and suggest a therapeutic target for cancer immunotherapy.

Cell death or survival: Insights into the role of mRNA translational control.

Cellular stress is an intrinsic part of cell physiology that underlines cell survival or death. The ability of mammalian cells to regulate global protein synthesis (aka translational control) represents a critical, yet underappreciated, layer of regulation during the stress response. Various cellular stress response pathways monitor conditions of cell growth and subsequently reshape the cellular translatome to optimize translational outputs. On the molecular level, such translational reprogramming involves an intricate network of interactions between translation machinery, RNA-binding proteins, mRNAs, and non-protein coding RNAs. In this review, we will discuss molecular mechanisms, signaling pathways, and targets of translational control that contribute to cellular adaptation to stress and to cell survival or death.

Stress granule-mediated sequestration of EGR1 mRNAs correlates with lomustine-induced cell death prevention.

Some chemotherapy drugs modulate the formation of stress granules (SGs), which are RNA-containing cytoplasmic foci contributing to stress response pathways. How SGs mechanistically contribute to pro-survival or pro-apoptotic functions must be better defined. The chemotherapy drug lomustine promotes SG formation by activating the stress-sensing eIF2α kinase HRI (encoded by the EIF2AK1 gene). Here, we applied a DNA microarray-based transcriptome analysis to determine the genes modulated by lomustine-induced stress and suggest roles for SGs in this process. We found that the expression of the pro-apoptotic EGR1 gene was specifically regulated in cells upon lomustine treatment. The appearance of EGR1-encoding mRNA in SGs correlated with a decrease in EGR1 mRNA translation. Specifically, EGR1 mRNA was sequestered to SGs upon lomustine treatment, probably preventing its ribosome translation and consequently limiting the degree of apoptosis. Our data support the model where SGs can selectively sequester specific mRNAs in a stress-specific manner, modulate their availability for translation, and thus determine the fate of a stressed cell.

Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids.

Nonvesicular extracellular RNAs (nv-exRNAs) constitute the majority of the extracellular RNAome, but little is known about their stability, function, and potential use as disease biomarkers. Herein, we measured the stability of several naked RNAs when incubated in human serum, urine, and cerebrospinal fluid (CSF). We identified extracellularly produced tRNA-derived small RNAs (tDRs) with half-lives of several hours in CSF. Contrary to widespread assumptions, these intrinsically stable small RNAs are full-length tRNAs containing broken phosphodiester bonds (i.e., nicked tRNAs). Standard molecular biology protocols, including phenol-based RNA extraction and heat, induce the artifactual denaturation of nicked tRNAs and the consequent in vitro production of tDRs. Broken bonds are roadblocks for reverse transcriptases, preventing amplification and/or sequencing of nicked tRNAs in their native state. To solve this, we performed enzymatic repair of nicked tRNAs purified under native conditions, harnessing the intrinsic activity of phage and bacterial tRNA repair systems. Enzymatic repair regenerated an RNase R-resistant tRNA-sized band in northern blot and enabled RT-PCR amplification of full-length tRNAs. We also separated nicked tRNAs from tDRs by chromatographic methods under native conditions, identifying nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids. Dissociation of nicked tRNAs produces single-stranded tDRs that can be spontaneously taken up by human epithelial cells, positioning stable nv-exRNAs as potentially relevant players in intercellular communication pathways.

Stress granules and neurodegeneration.

Recent advances suggest that the response of RNA metabolism to stress has an important role in the pathophysiology of neurodegenerative diseases, particularly amyotrophic lateral sclerosis, frontotemporal dementias and Alzheimer disease. RNA-binding proteins (RBPs) control the utilization of mRNA during stress, in part through the formation of membraneless organelles termed stress granules (SGs). These structures form through a process of liquid-liquid phase separation. Multiple biochemical pathways regulate SG biology. The major signalling pathways regulating SG formation include the mammalian target of rapamycin (mTOR)-eukaryotic translation initiation factor 4F (eIF4F) and eIF2α pathways, whereas the pathways regulating SG dispersion and removal are mediated by valosin-containing protein and the autolysosomal cascade. Post-translational modifications of RBPs also strongly contribute to the regulation of SGs. Evidence indicates that SGs are supposed to be transient structures, but the chronic stresses associated with ageing lead to chronic, persistent SGs that appear to act as a nidus for the aggregation of disease-related proteins. We suggest a model describing how intrinsic vulnerabilities within the cellular RNA metabolism might lead to the pathological aggregation of RBPs when SGs become persistent. This process might accelerate the pathophysiology of many neurodegenerative diseases and myopathies, and it suggests new targets for disease intervention.

Early rRNA processing is a stress-dependent regulatory event whose inhibition maintains nucleolar integrity.

The production of ribosomes is an energy-intensive process owing to the intricacy of these massive macromolecular machines. Each human ribosome contains 80 ribosomal proteins and four non-coding RNAs. Accurate assembly requires precise regulation of protein and RNA subunits. In response to stress, the integrated stress response (ISR) rapidly inhibits global translation. How rRNA is coordinately regulated with the rapid inhibition of ribosomal protein synthesis is not known. Here, we show that stress specifically inhibits the first step of rRNA processing. Unprocessed rRNA is stored within the nucleolus, and when stress resolves, it re-enters the ribosome biogenesis pathway. Retention of unprocessed rRNA within the nucleolus aids in the maintenance of this organelle. This response is independent of the ISR or inhibition of cellular translation but is independently regulated. Failure to coordinately control ribosomal protein translation and rRNA production results in nucleolar fragmentation. Our study unveils how the rapid translational shut-off in response to stress coordinates with rRNA synthesis production to maintain nucleolar integrity.

TIA1 potentiates tau phase separation and promotes generation of toxic oligomeric tau.

Tau protein plays an important role in the biology of stress granules and in the stress response of neurons, but the nature of these biochemical interactions is not known. Here we show that the interaction of tau with RNA and the RNA binding protein TIA1 is sufficient to drive phase separation of tau at physiological concentrations, without the requirement for artificial crowding agents such as polyethylene glycol (PEG). We further show that phase separation of tau in the presence of RNA and TIA1 generates abundant tau oligomers. Prior studies indicate that recombinant tau readily forms oligomers and fibrils in vitro in the presence of polyanionic agents, including RNA, but the resulting tau aggregates are not particularly toxic. We discover that tau oligomers generated during copartitioning with TIA1 are significantly more toxic than tau aggregates generated by incubation with RNA alone or phase-separated tau complexes generated by incubation with artificial crowding agents. This pathway identifies a potentially important source for generation of toxic tau oligomers in tau-related neurodegenerative diseases. Our results also reveal a general principle that phase-separated RBP droplets provide a vehicle for coassortment of selected proteins. Tau selectively copartitions with TIA1 under physiological conditions, emphasizing the importance of TIA1 for tau biology. Other RBPs, such as G3BP1, are able to copartition with tau, but this happens only in the presence of crowding agents. This type of selective mixing might provide a basis through which membraneless organelles bring together functionally relevant proteins to promote particular biological activities.

Rat Experimental Autoimmune Gastritis Model.

BACKGROUND: Autoimmune gastritis (AIG) is an autoimmune disease of the stomach characterized by the destruction of the oxyntic mucosa, which stops producing acid and becomes both functionally and morphologically atrophic. The pathogenic mechanisms behind the disease are still poorly understood. There is no early diagnosis and specific AIG therapy. To elucidate the pathogenesis of AIG, to search for early diagnostic markers, as well as to test new therapeutic approaches, an adequate and easily reproducible experimental model for autoimmune gastritis (EAG) is needed. Existing EAG models have some limitations, including slow development of signs, absence of advanced gastritis, irrational use of animals to obtain antigen. The aim was to find out whether it is possible to cause autoimmune gastritis similar to human disease in Wistar rats through immunization with a homologous gastric mucosa extract. METHODS: Wistar rats were immunized with gastric mucosa extract. Histology studies and evaluation of serological parameters were performed 56 and 91 days later. RESULTS: Destruction of oxyntic glands by infiltrating T lymphocytes were detected in rats on 56 and 91 days after initial immunization with gastric mucosa extract. Hyperplasia of enterochromaffin-like (ECL) cells was detected on the 91st day. Antral mucosa remained unchanged. CONCLUSION: Wistar rats, immunized with gastric mucosa extract, developed EAG similar to human AIG. The advantages of received EAG model are the ease of obtaining, the rapid development of oxyntic mucosa damage, which may progress to ECL cell hyperplasia.

A Solitary Stalled 80S Ribosome Prevents mRNA Recruitment to Stress Granules.

In response to stress stimuli, eukaryotic cells typically suppress protein synthesis. This leads to the release of mRNAs from polysomes, their condensation with RNA-binding proteins, and the formation of non-membrane-bound cytoplasmic compartments called stress granules (SGs). SGs contain 40S but generally lack 60S ribosomal subunits. It is known that cycloheximide, emetine, and anisomycin, the ribosome inhibitors that block the progression of 80S ribosomes along mRNA and stabilize polysomes, prevent SG assembly. Conversely, puromycin, which induces premature termination, releases mRNA from polysomes and stimulates the formation of SGs. The same effect is caused by some translation initiation inhibitors, which lead to polysome disassembly and the accumulation of mRNAs in the form of stalled 48S preinitiation complexes. Based on these and other data, it is believed that the trigger for SG formation is the presence of mRNA with extended ribosome-free segments, which tend to form condensates in the cell. In this study, we evaluated the ability of various small-molecule translation inhibitors to block or stimulate the assembly of SGs under conditions of severe oxidative stress induced by sodium arsenite. Contrary to expectations, we found that ribosome-targeting elongation inhibitors of a specific type, which arrest solitary 80S ribosomes at the beginning of the mRNA coding regions but do not interfere with all subsequent ribosomes in completing translation and leaving the transcripts (such as harringtonine, lactimidomycin, or T-2 toxin), completely prevent the formation of arsenite-induced SGs. These observations suggest that the presence of even a single 80S ribosome on mRNA is sufficient to prevent its recruitment into SGs, and the presence of extended ribosome-free regions of mRNA is not sufficient for SG formation. We propose that mRNA entry into SGs may be mediated by specific contacts between RNA-binding proteins and those regions on 40S subunits that remain inaccessible when ribosomes are associated.

Mammalian stress granules and P bodies at a glance.

Stress granules (SGs) and processing bodies (PBs) are membraneless ribonucleoprotein-based cellular compartments that assemble in response to stress. SGs and PBs form through liquid-liquid phase separation that is driven by high local concentrations of key proteins and RNAs, both of which dynamically shuttle between the granules and the cytoplasm. SGs uniquely contain certain translation initiation factors and PBs are uniquely enriched with factors related to mRNA degradation and decay, although recent analyses reveal much broader protein commonality between these granules. Despite detailed knowledge of their composition and dynamics, the function of SGs and PBs remains poorly understood. Both, however, contain mRNAs, implicating their assembly in the regulation of RNA metabolism. SGs may also serve as hubs that rewire signaling events during stress. By contrast, PBs may constitute RNA storage centers, independent of mRNA decay. The aberrant assembly or disassembly of these granules has pathological implications in cancer, viral infection and neurodegeneration. Here, we review the current concepts regarding the formation, composition, dynamics, function and involvement in disease of SGs and PBs.

Computer simulation of merotelic kinetochore-microtubule attachments: corona size is more important than other cell parameters.

The even chromosome segregation between daughter cells during mitosis is crucial for genome integrity and is mostly regulated by proper attachments of spindle microtubules to kinetochores. Abnormalities in this process can lead to chromosome mis-segregation and potentially result in severe developmental disorders such as aneuploidy and cancer. Merotelic attachments when tubulin microtubules captured by the kinetochore of one chromatid originate from both spindle poles are considered as one of the key molecular processes that cause such abnormalities. In this paper, we use computer modeling and the Monte Carlo approach to reveal the reasons for retaining merotelic attachments at the end of metaphase. To this end, we varied, in small increments, the basic cell parameters within ensembles of 100, 500, and 1000 virtual cells. The analysis of configurations that ensure the preservation of the largest fraction of merotelic attachments enabled us to conclude that only a change in the size of the kinetochore corona can significantly increase the number of merotelic attachments and the angle between the centromere axis and the spindle axis. The effect of the other changes in model parameters, if any, was steadily suppressed by the end of metaphase. In addition, our computer model was validated by successfully reproducing the results of third-party theoretical studies as well as some experimental observations. We also found that the orientation of chromosomes and the number of merotelic attachments do not have an explicit correlation with each other and within some limits can change independently.

Properties and biological impact of RNA G-quadruplexes: from order to turmoil and back.

Guanine-quadruplexes (G4s) are non-canonical four-stranded structures that can be formed in guanine (G) rich nucleic acid sequences. A great number of G-rich sequences capable of forming G4 structures have been described based on in vitro analysis, and evidence supporting their formation in live cells continues to accumulate. While formation of DNA G4s (dG4s) within chromatin in vivo has been supported by different chemical, imaging and genomic approaches, formation of RNA G4s (rG4s) in vivo remains a matter of discussion. Recent data support the dynamic nature of G4 formation in the transcriptome. Such dynamic fluctuation of rG4 folding-unfolding underpins the biological significance of these structures in the regulation of RNA metabolism. Moreover, rG4-mediated functions may ultimately be connected to mechanisms underlying disease pathologies and, potentially, provide novel options for therapeutics. In this framework, we will review the landscape of rG4s within the transcriptome, focus on their potential impact on biological processes, and consider an emerging connection of these functions in human health and disease.

eIF4G has intrinsic G-quadruplex binding activity that is required for tiRNA function.

As cells encounter adverse environmental conditions, such as hypoxia, oxidative stress or nutrient deprivation, they trigger stress response pathways to protect themselves until transient stresses have passed. Inhibition of translation is a key component of such cellular stress responses and mounting evidence has revealed the importance of a class of tRNA-derived small RNAs called tiRNAs in this process. The most potent of these small RNAs are those with the capability of assembling into tetrameric G-quadruplex (G4) structures. However, the mechanism by which these small RNAs inhibit translation has yet to be elucidated. Here we show that eIF4G, the major scaffolding protein in the translation initiation complex, directly binds G4s and this activity is required for tiRNA-mediated translation repression. Targeting of eIF4G results in an impairment of 40S ribosome scanning on mRNAs leading to the formation of eIF2α-independent stress granules. Our data reveals the mechanism by which tiRNAs inhibit translation and demonstrates novel activity for eIF4G in the regulation of translation.

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome.

A major proportion of extracellular RNAs (exRNAs) do not copurify with extracellular vesicles (EVs) and remain in ultracentrifugation supernatants of cell-conditioned medium or mammalian blood serum. However, little is known about exRNAs beyond EVs. We have previously shown that the composition of the nonvesicular exRNA fraction is highly biased toward specific tRNA-derived fragments capable of forming RNase-protecting dimers. To solve the problem of stability in exRNA analysis, we developed a method based on sequencing the size exclusion chromatography (SEC) fractions of nonvesicular extracellular samples treated with RNase inhibitors (RI). This method revealed dramatic compositional changes in exRNA population when enzymatic RNA degradation was inhibited. We demonstrated the presence of ribosomes and full-length tRNAs in cell-conditioned medium of a variety of mammalian cell lines. Their fragmentation generates some small RNAs that are highly resistant to degradation. The extracellular biogenesis of some of the most abundant exRNAs demonstrates that extracellular abundance is not a reliable input to estimate RNA secretion rates. Finally, we showed that chromatographic fractions containing extracellular ribosomes are probably not silent from an immunological perspective and could possibly be decoded as damage-associated molecular patterns.

RTCB Complex Regulates Stress-Induced tRNA Cleavage.

Under stress conditions, transfer RNAs (tRNAs) are cleaved by stress-responsive RNases such as angiogenin, generating tRNA-derived RNAs called tiRNAs. As tiRNAs contribute to cytoprotection through inhibition of translation and prevention of apoptosis, the regulation of tiRNA production is critical for cellular stress response. Here, we show that RTCB ligase complex (RTCB-LC), an RNA ligase complex involved in endoplasmic reticulum (ER) stress response and precursor tRNA splicing, negatively regulates stress-induced tiRNA production. Knockdown of RTCB significantly increased stress-induced tiRNA production, suggesting that RTCB-LC negatively regulates tiRNA production. Gel-purified tiRNAs were repaired to full-length tRNAs by RtcB in vitro, suggesting that RTCB-LC can generate full length tRNAs from tiRNAs. As RTCB-LC is inhibited under oxidative stress, we further investigated whether tiRNA production is promoted through the inhibition of RTCB-LC under oxidative stress. Although hydrogen peroxide (H2O2) itself did not induce tiRNA production, it rapidly boosted tiRNA production under the condition where stress-responsive RNases are activated. We propose a model of stress-induced tiRNA production consisting of two factors, a trigger and booster. This RTCB-LC-mediated boosting mechanism may contribute to the effective stress response in the cell.

RB49-like Bacteriophages Recognize O Antigens as One of the Alternative Primary Receptors.

The power of most of the enterobacterial O antigen types to provide robust protection against direct recognition of the cell surface by bacteriophage receptor-recognition proteins (RBP) has been recently recognized. The bacteriophages infecting O antigen producing strains of E. coli employ various strategies to tackle this nonspecific protection. T-even related phages, including RB49-like viruses, often have wide host ranges, being considered good candidates for use in phage therapy. However, the mechanisms by which these phages overcome the O antigen barrier remain unknown. We demonstrate here that RB49 and related phages Cognac49 and Whisky49 directly use certain types of O antigen as their primary receptors recognized by the virus long tail fibers (LTF) RBP gp38, so the O antigen becomes an attractant instead of an obstacle. Simultaneously to recognize multiple O antigen types, LTFs of each of these phages can bind to additional receptors, such as OmpA protein, enabling them to infect some rough strains of E. coli. We speculate that the mechanical force of the deployment of the short tail fibers (STF) triggered by the LTF binding to the O antigen or underneath of it, allows the receptor binding domains of STF to break through the O polysaccharide layer.