Studying the Regulation and Function of Biomolecular Condensates with In vitro Reconstitution

A central dogma in biochemistry is that a protein’s function depends on its fixed three-dimensional structure. However, bioinformatics analysis predicts that the majority of human proteins (~58%) contain both folded protein domains along with protein regions that are entirely devoid of any tertiary protein structures, referred to as intrinsically disordered protein regions (IDRs). Despite the strong association of proteins with significant IDRs to numerous diseases, most studies tend to overlook the potential function of IDRs and focus solely on their folded domain portions. Moreover, mutations within IDRs are hard to functionally interpret and become frequently neglected or annotated as variants of unknown significance. Given these observations, we need to understand better the function and biological role of IDRs in health and disease. Recently, IDRs have been shown to drive membrane-less RNA-protein granule formation. Similar to the separation of oil and water, specific proteins and nucleic acids can phase separate and form distinct membrane-less compartments (also referred to as condensates). While we have some understanding of the phase separation process, the biological regulation of phase separation and the functional consequences of condensates remains enigmatic. Here, I use biochemistry and biophysical approaches to study the regulation and function of IDR phase separation and extend these findings to disease implications. Beginning in chapter 3, I studied the phase separation of the C-terminal IDR of the fragile X mental retardation protein (FMRP). I demonstrated that post-translational modifications, including phosphorylation and methylation, regulate FMRP phase separation which correlates with in vitro translational inhibition. In chapter 4, I studied the protein interactions and co-phase separation properties between two interacting IDRs, from FMRP and CAPRIN1. I found that different phosphorylation patterns on each IDR can regulate their co-phase separation properties and modulate their impact on deadenylation and translational activity. In chapter 5, I showed that the solvent environment of a CAPRIN1 condensate can regulate deadenylation activity by CNOT7 within the condensate. Together, these results support the functional importance of IDR phase separation and also provide new methods to study condensates. Importantly, my findings provide a platform to understand how disease mutations within IDRs may perturb phase separation leading to disruption of biological function.

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