Characterization of RNA content in individual phase-separated coacervate microdroplets

Condensates formed by complex coacervation are hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of biological condensates and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and to which extend single condensates differ in their RNA composition. To address this, we developed an approach to read the RNA content from single synthetic and protein-based condensates using high-throughput sequencing. We find that certain RNAs efficiently accumulate in condensates. These RNAs are strongly enriched in sequence motifs which show high sequence similarity to short interspersed elements (SINEs). We observe similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution

Multifunctional Protein Materials and Microreactors using Low Complexity Domains as Molecular Adhesives

ISSN:1936-0851ISSN:1936-086

DEAD-box ATPases are global regulators of phase-separated organelles

The ability of proteins and nucleic acids to undergo liquid-liquid phase separation has recently emerged as an important molecular principle of how cells rapidly and reversibly compartmentalize their components into membrane-less organelles such as the nucleolus, processing bodies or stress granules; 1,2; . How the assembly and turnover of these organelles are controlled, and how these biological condensates selectively recruit or release components are poorly understood. Here we show that members of the large and highly abundant family of RNA-dependent DEAD-box ATPases (DDXs); 3; are regulators of RNA-containing phase-separated organelles in prokaryotes and eukaryotes. Using in vitro reconstitution and in vivo experiments, we demonstrate that DDXs promote phase separation in their ATP-bound form, whereas ATP hydrolysis induces compartment turnover and release of RNA. This mechanism of membrane-less organelle regulation reveals a principle of cellular organization that is conserved from bacteria to humans. Furthermore, we show that DDXs control RNA flux into and out of phase-separated organelles, and thus propose that a cellular network of dynamic, DDX-controlled compartments establishes biochemical reaction centres that provide cells with spatial and temporal control of various RNA-processing steps, which could regulate the composition and fate of ribonucleoprotein particles

Structural basis of histone H2A-H2B recognition by the essential chaperone FACT

Facilitates chromatin transcription (FACT) is a conserved histone chaperone that reorganizes nucleosomes and ensures chromatin integrity during DNA transcription, replication and repair(1-6). Key to the broad functions of FACT is its recognition of histones H2A-H2B (ref. 2). However, the structural basis for how histones H2A-H2B are recognized and how this integrates with the other functions of FACT, including the recognition of histones H3-H4 and other nuclear factors, is unknown. Here we reveal the crystal structure of the evolutionarily conserved FACT chaperone domain Spt16M from Chaetomium thermophilum, in complex with the H2A-H2B heterodimer. A novel 'U-turn' motif scaffolded onto a Rtt106-like module(7-10) embraces the alpha 1 helix of H2B. Biochemical and in vivo assays validate the structure and dissect the contribution of histone tails and H3-H4 towards Spt16M binding. Furthermore, we report the structure of the FACT heterodimerization domain that connects FACT to replicative polymerases. Our results show that Spt16M makes several interactions with histones, which we suggest allow the module to invade the nucleosome gradually and block the strongest interaction of H2B with DNA. FACT would thus enhance 'nucleosome breathing' by re-organizing the first 30 base pairs of nucleosomal histone-DNA contacts. Our snapshot of the engagement of the chaperone with H2A-H2B and the structures of all globular FACT domains enable the high-resolution analysis of the vital chaperoning functions of FACT, shedding light on how the complex promotes the activity of enzymes that require nucleosome reorganization

Spatial integration of transcription and splicing in a dedicated compartment sustains monogenic antigen expression in African trypanosomes

Highly selective gene expression is a key requirement for antigenic variation in several pathogens, allowing evasion of host immune responses and maintenance of persistent infections (1). African trypanosomes, parasites that cause lethal diseases in humans and livestock, employ an antigenic variation mechanism that involves monogenic antigen expression from a pool of >2600 antigen-coding genes (2). In other eukaryotes, the expression of individual genes can be enhanced by mechanisms involving the juxtaposition of otherwise distal chromosomal loci in the three-dimensional nuclear space (3–5). However, trypanosomes lack classical enhancer sequences or regulated transcription initiation (6,7). In this context, it has remained unclear how genome architecture contributes to monogenic transcription elongation and transcript processing. Here, we show that the single expressed antigen coding gene displays a specific inter-chromosomal interaction with a major mRNA splicing locus. Chromosome conformation capture (Hi-C) revealed a dynamic reconfiguration of this inter-chromosomal interaction upon activation of another antigen. Super-resolution microscopy showed the interaction to be heritable and splicing dependent. We find a specific association of the two genomic loci with the antigen exclusion complex, whereby VEX1 occupied the splicing locus and VEX2 the antigen coding locus. Following VEX2 depletion, loss of monogenic antigen expression was accompanied by increased interactions between previously silent antigen genes and the splicing locus. Our results reveal a mechanism to ensure monogenic expression, where antigen transcription and mRNA splicing occur in a specific nuclear compartment. These findings suggest a new means of post-transcriptional gene regulation