To Find and to Form: Cellular Strategies for Intracellular Target Search and Higher-Order Assembly

Abstract

Eukaryotic RNA-protein complexes have been widely reported to form membrane-less, higher-order assemblies inside cells under a range of conditions. How these structures contribute to the regulation of intracellular biochemistry remains poorly understood. Recent biophysical studies have revealed how phase-separation, a passive, thermodynamically driven process, can explain the assembly of such structures, referred to as condensates. This dissertation explores the relationship between macromolecular interactions that mediate the formation of dynamic condensates and the biochemical consequences of the resulting reorganization of the intracellular space. Organized into three parts, it implements and leverages new live-cell fluorescence microscopy approaches to visualize the formation of and localization of RNAs to condensates in real-time and at single-molecule resolution to address fundamental questions around intracellular biochemical regulation. First, the dissertation explores the RNA-sequence and protein translation-dependence of RNA localization to intracellular condensates called P-bodies. This work revealed that RNAs in P-bodies localize differently to the periphery or the core of these condensates depending on their translatability, and that stable RNA localization requires specific RNA-protein interactions. It next provides evidence for ubiquitous, proteome-wide, homomultimerization-driven phase-separation in response to osmotic volume fluctuations. These observations expand the molecular grammar of protein domains known to drive phase-separation, suggesting that a large fraction of the proteome may be poised to undergo rapid spatial reorganization upon small perturbations in intracellular molecular crowding. Additionally, these results provide possible explanations for previously reported features of osmotic stress response, by suggesting that hyperosmolarity-induced phase-separation of CPSF6 protein might provide a mechanistic basis for the widespread loss of premRNA cleavage activity under such conditions. These observations paint a new picture of the nature of the intracellular milieu, in which the organization of the intracellular space is inextricably linked with the macromolecular sequence of its constituents, where the concentration of individual molecular species can affect both its biochemical function and spatial organization. In the third part, the thesis discusses evidence that microRNA-induced silencing complexes may use a two-pronged strategy to search for mRNA targets inside the cell: on the one hand, transient binding and 3D search allow for rapid exploration; on the other hand, induced clustering of target mRNAs reduces the search space, such that these complexes can efficiently engage with their targets even when the concentration is limiting. Comparing the kinetics of individual microRNA-mRNA interactions in the cell across a range of mRNAs differing in the number of microRNA binding sites suggests that binding site number, a conserved feature of mRNAs, serves to both stabilize microRNA binding and promote AGO2-dependent clustering of mRNAs. This work refines an emerging paradigm in cell biology in which the intracellular space, far from being spatially homogeneous, is highly compartmentalized. Further, it demonstrates that such compartmentalization can be highly dynamic, and this dynamic organization is encoded by macromolecular sequence and biochemical activity. By applying single particle tracking to understand the assembly of intracellular condensate dynamics, this work opens up new ways for studying non-equilibrium phase separation and condensate formation in cells. Studying molecular association processes at single-molecule resolution in living cells represents a significant advance in quantitative cell biology by bridging single-molecule measurements in vitro and qualitative observations in vivo. This dissertation therefore advances the study of intracellular biochemistry by describing new methods and by applying them to uncover insights into the relationship between macromolecular sequence and subcellular organization.