Chronic social isolation causes severe psychological effects in humans, but their neural bases remain poorly understood. 2 weeks (but not 24 hr) of social isolation stress (SIS) caused multiple ...behavioral changes in mice and induced brain-wide upregulation of the neuropeptide tachykinin 2 (Tac2)/neurokinin B (NkB). Systemic administration of an Nk3R antagonist prevented virtually all of the behavioral effects of chronic SIS. Conversely, enhancing NkB expression and release phenocopied SIS in group-housed mice, promoting aggression and converting stimulus-locked defensive behaviors to persistent responses. Multiplexed analysis of Tac2/NkB function in multiple brain areas revealed dissociable, region-specific requirements for both the peptide and its receptor in different SIS-induced behavioral changes. Thus, Tac2 coordinates a pleiotropic brain state caused by SIS via a distributed mode of action. These data reveal the profound effects of prolonged social isolation on brain chemistry and function and suggest potential new therapeutic applications for Nk3R antagonists.
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•Chronic social isolation stress (SIS) causes a pervasive change in brain state•SIS broadly upregulates expression of Tac2/NkB in multiple brain regions•Tac2 upregulation is necessary and sufficient for behavioral influences of SIS•NkB acts locally in different brain areas to orchestrate behavioral effects of SIS
The Tac2 neuropeptide system orchestrates the complex behavioral effects of chronic social isolation stress by acting locally in multiple brain regions, suggesting the therapeutic potential of Nk3R antagonists for managing behavioral changes upon prolonged social isolation.
RNA, DNA, and protein molecules are highly organized within three-dimensional (3D) structures in the nucleus. Although RNA has been proposed to play a role in nuclear organization, exploring this has ...been challenging because existing methods cannot measure higher-order RNA and DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the spatial organization of RNA and DNA. These maps reveal higher-order RNA-chromatin structures associated with three major classes of nuclear function: RNA processing, heterochromatin assembly, and gene regulation. These data demonstrate that hundreds of ncRNAs form high-concentration territories throughout the nucleus, that specific RNAs are required to recruit various regulators into these territories, and that these RNAs can shape long-range DNA contacts, heterochromatin assembly, and gene expression. These results demonstrate a mechanism where RNAs form high-concentration territories, bind to diffusible regulators, and guide them into compartments to regulate essential nuclear functions.
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•RNA & DNA SPRITE comprehensively maps the spatial organization of RNA and DNA•Hundreds of ncRNAs form high-concentration territories throughout the nucleus•ncRNAs recruit diffusible RNA and protein regulators into precise 3D structures•ncRNA compartments can shape DNA contacts, heterochromatin, and gene expression
Mapping the proximity of RNAs to DNA and to other RNAs elucidates how nuclear non-coding RNAs serve as spatial organizers controlling processes underpinning gene regulation.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent ...need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.
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•NSP16 binds mRNA recognition domains of U1/U2 snRNAs and disrupts mRNA splicing•NSP1 binds in the mRNA entry channel of the ribosome to disrupt protein translation•NSP8 and NSP9 bind the signal recognition particle and disrupt protein trafficking•These disruptions of protein production suppress the interferon response to infection
SARS-CoV-2 proteins directly engage host RNAs to dysregulate essential steps of protein production and suppress the interferon response.
During pre-mRNA splicing, a central step in the expression and regulation of eukaryotic genes, the spliceosome selects splice sites for intron excision and exon ligation. In doing so, the spliceosome ...must distinguish optimal from suboptimal splice sites. At the catalytic stage of splicing, suboptimal splice sites are repressed by the DEAH-box ATPases Prp16 and Prp22. Here, using budding yeast, we show that these ATPases function further by enabling the spliceosome to search for and utilize alternative branch sites and 3′ splice sites. The ATPases facilitate this search by remodeling the splicing substrate to disengage candidate splice sites. Our data support a mechanism involving 3′ to 5′ translocation of the ATPases along substrate RNA and toward a candidate site, but, surprisingly, not across the site. Thus, our data implicate DEAH-box ATPases in acting at a distance by pulling substrate RNA from the catalytic core of the spliceosome.
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•The DEAH-box ATPase Prp16 enables alternative branch site selection•The DEAH-box ATPase Prp22 promotes alternative 3′ splice site selection•Prp16 and Prp22 both repress suboptimal sites by disengaging the splice sites•Prp16 and Prp22 remodel the substrate without translocating through their targets
Two DEAH-box ATPases enable the spliceosome to search for and utilize alternative branch sites and 3′ splice sites by disengaging suboptimal sites from the splicing machinery through a mechanism that suggests action at a distance by RNA pulling.
Although thousands of long non-coding RNAs (lncRNAs) are encoded in mammalian genomes, their mechanisms of action are poorly understood, in part because they are often expressed at lower levels than ...their proposed targets. One such lncRNA is Xist, which mediates chromosome-wide gene silencing on one of the two X chromosomes (X) to achieve gene expression balance between males and females. How a limited number of Xist molecules can mediate robust silencing of a much larger number of target genes while maintaining specificity exclusively to genes on the X within each cell is not well understood. Here, we show that Xist drives non-stoichiometric recruitment of the essential silencing protein SHARP (also known as SPEN) to amplify its abundance across the inactive X, including at regions not directly occupied by Xist. This amplification is achieved through concentration-dependent homotypic assemblies of SHARP on the X and is required for chromosome-wide silencing. Expression of Xist at higher levels leads to increased localization at autosomal regions, demonstrating that low levels of Xist are critical for ensuring its specificity to the X. We show that Xist (through SHARP) acts to suppress production of its own RNA which may act to constrain overall RNA levels and restrict its ability to spread beyond the X. Together, our results demonstrate a spatial amplification mechanism that allows Xist to achieve two essential but countervailing regulatory objectives: chromosome-wide gene silencing and specificity to the X. This suggests a more general mechanism by which other low-abundance lncRNAs could balance specificity to, and robust control of, their regulatory targets.
Many lncRNAs are thought to interact with the polycomb repressive complex 2 (PRC2) in order to regulate gene expression. A central example of this lncRNA–PRC2 paradigm in gene regulation is HOTAIR. ...In this issue of The EMBO Journal, a study (Portoso et al, ) reports that while HOTAIR binds PRC2 with high affinity, the complex itself is dispensable for HOTAIR‐mediated transcriptional silencing. This study raises important questions about the role of PRC2 interactions for lncRNA‐mediated functions and argues for a re‐evaluation of this lncRNA–PRC2 functional paradigm.
A new study showing Polycomb recruitment to be the consequence—not the cause—of target gene repression by lncRNA HOTAIR questions the role of lncRNA–PRC2 complexes in transcriptional silencing.
In response to intracellular signals in Gram--negative bacteria, translational riboswitches--commonly embedded in messenger RNAs (mRNAs)-regulate gene expression through inhibition of translation ...initiation. It is generally thought that this regulation originates from occlusion of the Shine-Dalgarno (SD) sequence upon ligand binding; however, little direct evidence exists. Here we develop Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) to investigate the ligand-dependent accessibility of the SD sequence of an mRNA hosting the 7-aminomethyl-7-deazaguanine (preQ1)-sensing riboswitch. Spike train analysis reveals that individual mRNA molecules alternate between two conformational states, distinguished by 'bursts' of probe binding associated with increased SD sequence accessibility. Addition of preQ1 decreases the lifetime of the SD's high-accessibility (bursting) state and prolongs the time between bursts. In addition, ligand-jump experiments reveal imperfect riboswitching of single mRNA molecules. Such complex ligand sensing by individual mRNA molecules rationalizes the nuanced ligand response observed during bulk mRNA translation.
•Emergence was conceptualized as serial dormancy release and germination processes.•Dormancy release and germination were modeled as logistic distributions.•Avena fatua field emergence data from a ...semiarid region were analyzed.•A genetic algorithm was adopted to solve the parameters estimation problem.•The proposed approach yield better predictions than NLR and ANN models.
Avena fatua is a cosmopolite weed species which produces severe yield losses in small-grain production systems in temperate and semiarid climates. In the semiarid region of Argentina, A. fatua field emergence patterns show great year-to-year variability mainly due to the effect of highly unpredictable precipitation regimes as well as a complex seedbank dormancy behavior regulated by both, genetic and environmental factors. Previously developed models for the same agroecological system based on Non-Linear Regression techniques (NLR) and Artificial Neural Networks (ANN) were either unable to accurately predict field emergence or lacked explanatory power. The main objective of the present work is to develop a simple (i.e. parsimonious) model for A. fatua field emergence prediction for the semiarid region of Argentina based on the disaggregation of the dormancy release phase from the germination/pre-emergence growth processes, using easy accessible soil microclimate derived indices as input variables and observed cumulative field emergence data as output variable. The parsimony and predictive capability of the newly developed model were compared with NLR and ANN approaches developed by the same authors for the same agroecological system. Specifically, dormancy release was modeled as a logistic function of an after-ripening thermal-time index while germination/pre-emergence growth was represented by a logistic distribution of hydrothermal-time accumulation. A total of 528 input/output data pairs corresponding to 11 years of data collection were used in this study. Due to its implementation simplicity and good convergence features, a Genetic Algorithm (GA) was adopted to solve the resulting optimization problem consisting on the minimization of the Mean Square Error (MSE) between training data and experimentally obtained field emergence data. The newly developed GA based approach resulted in a significantly more parsimonious model (BIC=−1.54) compared to ANN (BIC=−1.36) and NLR (BIC=−1.32) models. Model evaluation with independent data also showed a better predictive capacity of the GA approach (RMSE=0.07) compared to NLR (RMSE=0.19) and ANN (RMSE=0.11) alternatives. These outcomes suggest the potential applicability of the proposed predictive tool in weed management decision support systems design.
The spliceosome is a dynamic ribonucleoprotein (RNP) machine that catalyzes the removal of introns during the two transesterification steps of eukaryotic pre-mRNA splicing. Here we used ...single-molecule fluorescence resonance energy transfer to monitor the distance of the 5' splice site (5' SS) and branch point (BP) of pre-mRNA in affinity-purified spliceosomes stalled by a mutation in the DExD/H-box helicase Prp2 immediately before the first splicing step. Addition of recombinant Prp2 together with NTP and protein cofactor Spp2 rearranges the spliceosome-substrate complex to reversibly explore conformations with proximal 5' SS and BP that accommodate chemistry. Addition of Cwc25, a small heat-stable splicing factor, then strongly biases this equilibrium toward the proximal conformation, promoting efficient first-step splicing. The spliceosome thus functions as a biased Brownian ratchet machine where a helicase unlocks thermal fluctuations subsequently rectified by a cofactor 'pawl', a principle possibly widespread among the many helicase-driven RNPs.
Polycomb repressive complex 2 (PRC2) is reported to bind to many RNAs and has become a central player in reports of how long non-coding RNAs (lncRNAs) regulate gene expression. Yet, there is a ...growing discrepancy between the biochemical evidence supporting specific lncRNA-PRC2 interactions and functional evidence demonstrating that PRC2 is often dispensable for lncRNA function. Here, we revisit the evidence supporting RNA binding by PRC2 and show that many reported interactions may not occur in vivo. Using denaturing purification of in vivo crosslinked RNA-protein complexes in human and mouse cell lines, we observe a loss of detectable RNA binding to PRC2 and chromatin-associated proteins previously reported to bind RNA (CTCF, YY1, and others), despite accurately mapping bona fide RNA-binding sites across others (SPEN, TET2, and others). Taken together, these results argue for a critical re-evaluation of the broad role of RNA binding to orchestrate various chromatin regulatory mechanisms.
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•Many previously detected PRC2-RNA interactions do not occur in vivo•Denaturing purifications eliminate PRC2-RNA interactions but retain known interactions•Other reported chromatin regulators (CTCF and YY1) do not appear to bind RNA in vivo•Broad role for RNA binding in chromatin regulation needs to be re-evaluated
This work by Guo et al. challenges widespread reports that PRC2 and other chromatin proteins bind to RNA to control the regulation of gene expression. It highlights the discrepancy between biochemical and functional evidence and suggests that reports of a broad role for protein-RNA binding in controlling chromatin regulatory mechanisms should be re-evaluated.
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