Abstract
RNA granules consist of membrane-less RNA-protein assemblies and contain dynamic liquid-like shells and stable solid-like cores, which are thought to function in numerous processes in mRNA sorting and translational regulation. However, how these distinct substructures are formed, whether they are assembled by different scaffolds, and whether different RNA granule scaffolds induce these different substructures remains unknown. Here, using fluorescence microscopy-based morphological and molecular-dynamics analyses, we demonstrate that RNA granule scaffold proteins (scaffolds) can be largely classified into two groups, liquid and solid types, which induce the formation of liquid-like and solid-like granules, respectively, when expressed separately in cultured cells. We found that when co-expressed, the liquid-type and solid-type scaffolds combine and form liquid- and solid-like substructures in the same granules, respectively. The combination of the different types of scaffolds reduced the immobile fractions of the solid-type scaffolds and their dose-dependent ability to decrease nascent polypeptides in granules, but had little effect on the dynamics of the liquid-type scaffolds or their dose-dependent ability to increase nascent polypeptides in granules. These results suggest that solid- and liquid-type scaffolds form different substructures in RNA granules and differentially affect each other. Our findings provide detailed insight into the assembly mechanism and distinct dynamics and functions of core and shell substructures in RNA granules.
Highlights
RNA granules consist of membrane-less RNA–protein assemblies and contain dynamic liquid-like shells and stable solid-like cores, which are thought to function in numerous processes in mRNA sorting and translational regulation
By expressing RNA granule scaffolds in cells, we demonstrated that solid core-like R-structures and liquid shell-like S-structures were formed by distinct scaffolds, as judged by morphological analysis and dynamics analysis
R-granules were induced by T-cell intracellular antigen 1 (TIA-1), TIA-1–related protein (TIAR), fragile X mental retardation 1 (FMR1), fused in sarcoma (FUS), and Pumilio1, whereas S-granules were induced by GTPase–activating protein SH3 domain-binding protein 1 (G3BP1), RNA granule protein 105 (RNG105), and TAR DNA– binding protein 43 (TDP-43)
Summary
Many RNA granule-associated proteins have been identified, among which several proteins are known to induce RNA granule assembly when expressed in cells and are designated as scaffolds [2, 17]. RNG105, G3BP1, and TDP-43 blew out of the permeabilized cells, but they were not observed as foci (Fig. 2D and Movie S6), suggesting that the granules formed by the S-scaffolds were not purifiable, which contrasted with the R-granules. R-scaffolds retained the properties observed when expressed alone, even in the presence of S-scaffolds as follows: FMR1- and Pumilio1-induced R-substructures were resistant to cell permeabilization and retained their R-texture (Fig. 6A and Movie S13). Before analyzing the effects of co-expression of the scaffolds, we first measured the amount of nascent polypeptides (puro intensity) in cells expressing Pumilio, FUS, RNG105, or G3BP1 alone using the ribopuromycilation analysis (Fig. 8, A and B). In contrast to the R-scaffolds, S-scaffolds, i.e. RNG105 and G3BP1, dose-dependently increased puro intensity in the granules, whereas the cytoplasmic puro intensity remained lower than that in control cells (Fig. 8, C and D). These results suggested that the combination with S-granules influenced the physiological properties as well as dynamics of R-granules
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