RNA-Mediated Feedback Control of Transcriptional Condensates.
Ontology highlight
ABSTRACT:
SUBMITTER: Henninger JE
PROVIDER: S-EPMC8128340 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:RNA-binding proteins (RBPs) facilitate post-transcriptional control of eukaryotic gene expression at multiple levels. The RBP tristetraprolin (TTP/Zfp36) is a signal-induced phosphorylated anti-inflammatory protein guiding unstable mRNAs of pro-inflammatory proteins for degradation and preventing translation. Using iCLIP, we have identified numerous mRNA targets bound by wild-type TTP and by a non-MK2-phosphorylatable TTP mutant (TTP-AA) in 1 h LPS-stimulated macrophages and correlated their interaction with TTP to changes at the level of mRNA abundance and translation in a transcriptome-wide manner. The close similarity of the transcriptomes of TTP-deficient and TTP-expressing macrophages upon short LPS stimulation suggested an effective inactivation of TTP by MK2, whereas retained RNA-binding capacity of TTP-AA to 3'UTRs caused profound changes in the transcriptome and translatome, altered NF-κB-activation and induced cell death. Increased TTP binding to the 3'UTR of feedback inhibitor mRNAs, such as Ier3, Dusp1 or Tnfaip3, in the absence of MK2-dependent TTP neutralization resulted in a strong reduction of their protein synthesis contributing to the deregulation of the NF-κB-signaling pathway. Taken together, our study uncovers a role of TTP as a suppressor of feedback inhibitors of inflammation and highlights the importance of fine-tuned TTP activity-regulation by MK2 in order to control the pro-inflammatory response.
| S-EPMC5009735 | biostudies-literature
Project description:Designer RNA scaffolds constitute a promising tool for synthetic biology, as they can be genetically expressed to perform specific functions in vivo such as scaffolding enzymatic cascades and regulating gene expression through CRISPR-dCas9 applications. RNA origami is a recently developed RNA design approach that allows construction of large RNA nanostructures that can position aptamer motifs to spatially organize other molecules, including proteins. However, it is still not fully understood how positioning multiple aptamers on a scaffold and the orientation of a scaffold affects functional properties. Here, we investigate fusions of single-guide RNAs and RNA origami scaffolds (termed sgRNAO) capable of recruiting activating domains for control of gene expression in yeast. Using MS2 and PP7 as orthogonal protein-binding aptamers, we observe a gradual increase in transcriptional activation for up to four aptamers. We demonstrate that different aptamer positions on a scaffold and scaffold orientation affect transcriptional activation. Finally, sgRNAOs are used to regulate expression of enzymes of the violacein biosynthesis pathway to control metabolic flux. The integration of RNA origami nanostructures at promoter sites achieved here, can in the future be expanded by the addition of functional motifs such as riboswitches, ribozymes and sensor elements to allow for complex gene regulation.
| S-EPMC9262615 | biostudies-literature
Project description:Biomolecular condensates assembled through liquid-liquid phase separation (LLPS) of proteins and RNAs are currently recognized to play an important role in cellular organization. Their assembly depends on the formation of a network of transient, multivalent interactions between flexible scaffold biomolecules. Understanding how protein and RNA sequences determine these interactions and ultimately regulate the phase separation is an open key challenge. Recent in vitro studies have revealed that arginine and lysine residues, which are enriched in most cellular condensates, have markedly distinct propensities to drive the LLPS of protein/RNA mixtures. Here, we employ explicit-solvent atomistic molecular dynamics simulations to shed light on the microscopic origin of this difference by investigating mixtures of polyU oligonucleotides with either polyR/polyK peptides. In agreement with experiments, our simulations indicate that arginine has a higher affinity for polyU than lysine both in highly diluted conditions and in concentrated solutions with a biomolecular density comparable to cellular condensate. The analysis of intermolecular contacts suggests that this differential behavior is due to the propensity of arginine side chains to simultaneously form a higher number of specific interactions with oligonucleotides, including hydrogen bonds and stacking interactions. Our results provide a molecular description of how the multivalency of the guanidinium group enables the coordination of multiple RNA groups by a single arginine residue, thus ultimately stabilizing protein/RNA condensates.
| S-EPMC8197427 | biostudies-literature
Project description:The human pathogen Candida albicans can assume either of two distinct cell types, designated "white" and "opaque." Each cell type is maintained for many generations; switching between them is rare and stochastic, and occurs without any known changes in the nucleotide sequence of the genome. The two cell types differ dramatically in cell shape, colony appearance, mating competence, and virulence properties. In this work, we investigate the transcriptional circuitry that specifies the two cell types and controls the switching between them. First, we identify two new transcriptional regulators of white-opaque switching, Czf1 and white-opaque regulator 2 (Wor2). Analysis of a large set of double mutants and ectopic expression strains revealed genetic relationships between CZF1, WOR2, and two previously identified regulators of white-opaque switching, WOR1 and EFG1. Using chromatin immunoprecipitation, we show that Wor1 binds the intergenic regions upstream of the genes encoding three additional transcriptional regulators of white-opaque switching (CZF1, EFG1, and WOR2), and also occupies the promoters of numerous white- and opaque-enriched genes. Based on these interactions, we have placed these four genes in a circuit controlling white-opaque switching whose topology is a network of positive feedback loops, with the master regulator gene WOR1 occupying a central position. Our observations indicate that a key role of the interlocking feedback loop network is to stably maintain each epigenetic state through many cell divisions.
| S-EPMC1976629 | biostudies-literature
Project description:MicroRNAs (miRNAs) are small noncoding RNAs that regulate complex gene expression networks in eukaryotic cells. Because of their unique expression patterns, miRNAs are potential molecular markers for specific cell states. Although a system capable of imaging miRNA in living cells is needed to visually detect miRNA expression, very few fluorescence signal-on sensors that respond to expression of target miRNA (miR-ON sensors) are available. Here we report an miR-ON sensor containing a bidirectional promoter-driven Csy4 endoribonuclease and green fluorescent protein, ZsGreen1, for live-cell imaging of miRNAs with post-transcriptional feedback control. Csy4-assisted miR-ON (Csy4-miR-ON) sensors generate negligible background but respond sensitively to target miRNAs, allowing high-contrast fluorescence detection of miRNAs in various human cells. We show that Csy4-miR-ON sensors enabled imaging of various miRNAs, including miR-21, miR-302a, and miR-133, in vitro as well as in vivo. This robust tool can be used to evaluate miRNA expression in diverse biological and medical applications.
| S-EPMC8479275 | biostudies-literature
Project description:Enhancers are DNA elements that are bound by transcription factors (TFs), which recruit coactivators and the transcriptional machinery to genes. Phase-separated condensates of TFs and coactivators have been implicated in assembling the transcription machinery at particular enhancers, yet the role of DNA sequence in this process has not been explored. We show that DNA sequences encoding TF binding site number, density, and affinity above sharply defined thresholds drive condensation of TFs and coactivators. A combination of specific structured (TF-DNA) and weak multivalent (TF-coactivator) interactions allows for condensates to form at particular genomic loci determined by the DNA sequence and the complement of expressed TFs. DNA features found to drive condensation promote enhancer activity and transcription in cells. Our study provides a framework to understand how the genome can scaffold transcriptional condensates at specific loci and how the universal phenomenon of phase separation might regulate this process.
| S-EPMC6690378 | biostudies-literature
Project description:In live cells, phase separation is thought to organize macromolecules into membraneless structures known as biomolecular condensates. Here, we reconstituted transcription in condensates from purified mitochondrial components using optimized in vitro reaction conditions to probe the structure-function relationships of biomolecular condensates. We find that the core components of the mt-transcription machinery form multiphasic, viscoelastic condensates in vitro. Strikingly, the rates of condensate-mediated transcription are substantially lower than in solution. The condensate-mediated decrease in transcriptional rates is associated with the formation of vesicle-like structures that are driven by the production and accumulation of RNA during transcription. The generation of RNA alters the global phase behavior and organization of transcription components within condensates. Coarse-grained simulations of mesoscale structures at equilibrium show that the components stably assemble into multiphasic condensates and that the vesicles formed in vitro are the result of dynamical arrest. Overall, our findings illustrate the complex phase behavior of transcribing, multicomponent condensates, and they highlight the intimate, bidirectional interplay of structure and function in transcriptional condensates.
| S-EPMC9565167 | biostudies-literature
Project description:Intracellular aggregation of repeat expanded RNA has been implicated in many neurological disorders. Here, we study the role of biomolecular condensates on irreversible RNA clustering. We find that physiologically relevant and disease-associated repeat RNAs spontaneously undergo an age-dependent percolation transition inside multi-component protein-nucleic acid condensates to form nanoscale clusters. Homotypic RNA clusters drive the emergence of multiphasic condensate structures with an RNA-rich solid core surrounded by an RNA-depleted fluid shell. The timescale of the RNA clustering, which drives a liquid-to-solid transition of biomolecular condensates, is determined by the sequence features, stability of RNA secondary structure, and repeat length. Importantly, G3BP1, the core scaffold of stress granules, introduces heterotypic buffering to homotypic RNA-RNA interactions and impedes intra-condensate RNA clustering in an ATP-independent manner. Our work suggests that biomolecular condensates can act as sites for RNA aggregation. It also highlights the functional role of RNA-binding proteins in suppressing aberrant RNA phase transitions.
| S-EPMC11195159 | biostudies-literature
Project description:Expansions of amino acid repeats occur in >20 inherited human disorders, and many occur in intrinsically disordered regions (IDRs) of transcription factors (TFs). Such diseases are associated with protein aggregation, but the contribution of aggregates to pathology has been controversial. Here, we report that alanine repeat expansions in the HOXD13 TF, which cause hereditary synpolydactyly in humans, alter its phase separation capacity and its capacity to co-condense with transcriptional co-activators. HOXD13 repeat expansions perturb the composition of HOXD13-containing condensates in vitro and in vivo and alter the transcriptional program in a cell-specific manner in a mouse model of synpolydactyly. Disease-associated repeat expansions in other TFs (HOXA13, RUNX2, and TBP) were similarly found to alter their phase separation. These results suggest that unblending of transcriptional condensates may underlie human pathologies. We present a molecular classification of TF IDRs, which provides a framework to dissect TF function in diseases associated with transcriptional dysregulation.
| S-EPMC7261253 | biostudies-literature
Project description:Enhancers are DNA sequences at a long genomic distance from target genes. Recent experiments suggest that enhancers are anchored to the surfaces of condensates of transcription machinery and that the loop extrusion process enhances the transcription level of their target genes. Here, we theoretically study the polymer dynamics driven by the loop extrusion of the linker DNA between an enhancer and the promoter of its target gene to calculate the contact probability of the promoter to the transcription machinery in the condensate. Our theory predicts that when the loop extrusion process is active, the contact probability increases with increasing linker DNA length. This finding reflects the fact that the relaxation time, with which the promoter stays in proximity to the surface of the transcriptional condensate, increases as the length of the linker DNA increases. This contrasts the equilibrium case for which the contact probability between the promoter and the transcription machineries is smaller for longer linker DNA lengths.
| S-EPMC8136786 | biostudies-literature