ABSTRACT
Cellular homeostasis requires the robust control of biomolecule concentrations, but how do millions of mRNAs coordinate their stoichiometries in the face of dynamic translational changes? Here, we identified a two-tiered mechanism controlling mRNA:mRNA and mRNA:protein stoichiometries where mRNAs super-assemble into condensates with buffering capacity and sorting selectivity through phase-transition mechanisms. Using C. elegans oogenesis arrest as a model, we investigated the transcriptome cytosolic reorganization through the sequencing of RNA super-assemblies coupled with single mRNA imaging. Tightly repressed mRNAs self-assembled into same-sequence nanoclusters that further co-assembled into multiphase condensates. mRNA self-sorting was concentration dependent, providing a self-buffering mechanism that is selective to sequence identity and controls mRNA:mRNA stoichiometries. The cooperative sharing of limiting translation repressors between clustered mRNAs prevented the disruption of mRNA:repressor stoichiometries in the cytosol. Robust control of mRNA:mRNA and mRNA:protein stoichiometries emerges from mRNA self-demixing and cooperative super-assembly into multiphase multiscale condensates with dynamic storage capacity.
Subject(s)
Biomolecular Condensates , Caenorhabditis elegans , RNA, Messenger , Animals , Caenorhabditis elegans/cytology , Caenorhabditis elegans/metabolism , Oogenesis , Protein Biosynthesis , RNA Transport , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Proteins/chemistry , Proteins/metabolism , Biomolecular Condensates/chemistry , Biomolecular Condensates/metabolismABSTRACT
Stress granules (SGs) are macromolecular assemblies induced by stress and composed of proteins and mRNAs stalled in translation initiation. SGs play an important role in the response to stress and in the modulation of signaling pathways. Furthermore, these structures are related to the pathological ribonucleoprotein (RNP) aggregates found in neurodegenerative disease contexts, highlighting the need to understand how they are formed and recycled in normal and pathological contexts. Although genetically tractable multicellular organisms have been key in identifying modifiers of RNP aggregate toxicity, in vivo analysis of SG properties and regulation has lagged behind, largely due to the difficulty of detecting SG from images of intact tissues. Here, we describe the object detector software Obj.MPP and show how it overcomes the limits of classical object analyzers to extract the properties of SGs from wide-field and confocal images of Caenorhabditis elegans and Drosophila tissues, respectively. We demonstrate that Obj.MPP enables the identification of genes modulating the assembly of endogenous and pathological SGs, and thus that it will be useful in the context of future genetic screens and in vivo studies.