Cochard A

Bio: Cochard A is an academic researcher from École Normale Supérieure. The author has contributed to research in topics: Stress granule & Nucleolus. The author has an hindex of 1, co-authored 1 publications receiving 3 citations. Previous affiliations of Cochard A include University of Paris.

RNA at the surface of phase-separated condensates impacts their size and number

Abstract: Membrane-less organelles, by localizing and regulating complex biochemical reactions, are ubiquitous functional subunits of intracellular organization. They include a variety of nuclear and cytoplasmic ribonucleoprotein (RNP) condensates, such as nucleoli, P-bodies, germ granules and stress granules. While is it now recognized that specific RNA and protein families are critical for the biogenesis of RNP condensates, how these molecular constituents determine condensate size and morphology is unknown. To circumvent the biochemical complexity of endogenous RNP condensates, the use of programmable tools to reconstitute condensate formation with minimal constituents can be instrumental. Here we report a methodology to form RNA-containing condensates in living cells with controlled RNA and protein composition. Our bioengineered condensates are made of ArtiGranule scaffolds undergoing liquid-liquid phase separation in cells and programmed to specifically recruit a unique RNA species. We found that RNAs localized on condensate surface, either as isolated RNA molecules or as a homogenous corona of RNA molecules around the condensate. This simplified system allowed us to demonstrate that the size of the condensates scales with RNA surface density, the higher the RNA density is, the smaller and more frequent the condensates are. Our observations suggest a mechanism based on physical constraints, provided by RNAs localized on condensate surface, that limit condensate growth and coalescence.

Size conservation emerges spontaneously in biomolecular condensates formed by scaffolds and surfactant clients.

Abstract: Biomolecular condensates are liquid-like membraneless compartments that contribute to the spatiotemporal organization of proteins, RNA, and other biomolecules inside cells. Some membraneless compartments, such as nucleoli, are dispersed as different condensates that do not grow beyond a certain size, or do not present coalescence over time. In this work, using a minimal protein model, we show that phase separation of binary mixtures of scaffolds and low-valency clients that can act as surfactants-i.e., that significantly reduce the droplet surface tension-can yield either a single drop or multiple droplets that conserve their sizes on long timescales (herein 'multidroplet size-conserved' scenario'), depending on the scaffold to client ratio. Our simulations demonstrate that protein connectivity and condensate surface tension regulate the balance between these two scenarios. The multidroplet size-conserved scenario spontaneously arises at increasing surfactant-to-scaffold concentrations, when the interfacial penalty for creating small liquid droplets is sufficiently reduced by the surfactant proteins that are preferentially located at the interface. In contrast, low surfactant-to-scaffold concentrations enable continuous growth and fusion of droplets without restrictions. Overall, our work proposes one thermodynamic mechanism to help rationalize how size-conserved coexisting condensates can persist inside cells-shedding light on the roles of protein connectivity, binding affinity, and droplet composition in this process.

Three-dimensional genome organization via triplex-forming RNAs.

Abstract: An increasing number of long noncoding RNAs (lncRNAs) have been proposed to act as nuclear organization factors during interphase. Direct RNA-DNA interactions can be achieved by the formation of triplex helix structures where a single-stranded RNA molecule hybridizes by complementarity into the major groove of double-stranded DNA. However, whether and how these direct RNA-DNA associations influence genome structure in interphase chromosomes remain poorly understood. Here we theorize that RNA organizes the genome in space via a triplex-forming mechanism. To test this theory, we apply a computational modeling approach of chromosomes that combines restraint-based modeling with polymer physics. Our models suggest that colocalization of triplex hotspots targeted by lncRNAs could contribute to large-scale chromosome compartmentalization cooperating, rather than competing, with architectural transcription factors such as CTCF.

Mesoscale structure–function relationships in mitochondrial transcriptional condensates

Abstract: Significance Many droplet-like cellular structures lack membranes and are referred to as biomolecular condensates. One fundamental process associated with condensates is gene expression, in which the protein transcription machinery generates RNA de novo from a DNA template. We used mitochondrial transcription as a model system to ask how the structure of a condensate is influenced by the transcription that occurs within it. We were able to reconstitute mitochondrial transcription under condensate-forming conditions and show that the presence of the condensate reduces the efficiency of transcription. In turn, we also found that the production of RNA alters the structure of the condensate. These results reveal that structure and function are inherently coupled in transcriptional condensates.

Nucleobase Clustering Contributes to the Formation and Hollowing of Repeat-Expansion RNA Condensate

Abstract: RNA molecules with repeat expansion sequences can phase separate into gel-like condensate, which could lead to neurodegenerative diseases. Here, we report that, in the presence of Mg2+, RNA molecules containing 20× CAG repeats self-assemble into three morphologically distinct droplets. Using hyperspectral stimulated Raman microscopy, we show that RNA phase separation is accompanied by the clustering of nucleobases while forfeiting the canonical base-paired structure. As the RNA/Mg2+ ratio increases, the RNA droplets first expand and then shrink to adopt hollow vesicle-like structures. Significantly, for both large and vesicle-like RNA droplets, the nucleobase-clustered structure is more prominent at the rim, suggesting a continuously hardening process. This mechanism may be implicated in the general aging processes of RNA-containing membrane-less organelles.