Intrinsically Disordered Regions Can Contribute Promiscuous Interactions to RNP Granule Assembly

Abstract

Eukaryotic cells contain large RNA-protein assemblies referred to as RNP granules, whose assembly is promoted by both traditional protein interactions and intrinsically disordered protein domains. Using RNP granules as an example, we provide evidence for an assembly mechanism of large cellular structures wherein specific protein-protein or protein-RNA interactions act together with promiscuous interactions of intrinsically disordered regions (IDRs). This synergistic assembly mechanism illuminates RNP granule assembly and explains why many components of RNP granules, and other large dynamic assemblies, contain IDRs linked to specific protein-protein or protein-RNA interaction modules. We suggest assemblies based on combinations of specific interactions and promiscuous IDRs are common features of eukaryotic cells.

Keywords: Dhh1; P-body; RNP granule; intrinsically disordered; phase separation.

Figure 1. Competitor Proteins Disrupt IDR-Driven Phase Separations

A) Domain structure of hnRNP A1, FUS, and eIF4GII. B) Fluorescent and bright-field microscopy images of phase separated droplets formed at 37.5 mMNaCl by 25 µM SNAP-hnRNP A1_IDR or 2.1 µM SNAP-hnRNP A1_IDR with the indicated concentrations of BSA. Images each independently scaled. C) Box and whisker plot of structure size for SNAP-hnRNP A1_Δhexa from (B), significance calculated with Welch’s t test for unequal size and variance. Whiskers indicate distribution limits, excluding outliers. D) Quantification of the intensity of all structures between areas of 3 µm and 4 µm for hnRNPA1_Δhexa from (B). Subsets of droplets have roughly equal distributions of size (inset).

Figure 2. Disruption of LLPS by Other Proteins Is a General Phenomenon

A) Phase-separated droplets formed at 37.5 mM NaCl by SNAP-hnRNP A1_IDR, SNAP-FUS_IDR, SNAP-eIF4GII_IDR in the absence or presence of 100 mg/mL BSA, lysozyme, or RNase A. Images each independently scaled. B) Phase-separated droplets formed at 37.5 mM NaCl by SNAP-hnRNP A1_Δhexa, SNAP-hnRNP A1_IDR, and SNAP-FUS_IDR in the absence or presence of ~10 mg/mL yeast lysate. Pairs of fluorescent images are scaled to the 0 mg/mL image.

Figure 3. Globular Proteins Are Recruited to IDR-Driven LLPS Droplets

A) Phase-separated droplets formed at 37.5 mM NaCl by 25 µM SNAP-hnRNP A1_Δhexa and 500 nM FITC-labeled BSA or FITC-labeled lysozyme. Images each independently scaled. (B) Phase-separated droplets formed by SNAP-eIF4GII_IDR (35 µM), SNAP-hnRNP A1_IDR (5.25 µM), or SNAP-FUS_IDR (5 µM) in the presence of either 10 nM FITC-BSA or 100 nM FITC-lysozyme. Images each independently scaled.

Figure 4. IDRs Enhance LLPS of PTB plus RNA in the Presence of BSA

A) Phase-separated droplets formed by SNAP-PTB and RNA in the presence or absence of 100 mg/mL BSA, Ficoll, or PEG. B) Phase-separated droplets of 4µM SNAP-PTB, SNAP-PTB-FUS_IDR, or SNAP-PTB-Pub1_IDR, plus 0.32 µM RNA assemblies in the presence or absence of 100 mg/mL BSA. C) Quantification of assembly area for (B) with arbitrary units. Welch’s t test.

Figure 5. IDRs Are Neither Sufficient nor Required for P-Body Localization

A) Domain structures of the yeast proteins Dhh1, Lsm4, Ccr4, and Pop2. B) Dhh1-GFP variant fusions were expressed in Edc3-mCherry expressing yeast. P-bodies were visualized by fluorescence microscopy after 10 min glucose deprivation. C) Quantification of the percentage of P-bodies that exhibited colocalization with the expressed fusion protein. GFP was fused to the N terminus of Dhh1, Ccr4, and Pop2 variants. Variants were co-transformed with Edc3-mCherry. mCherry was fused to the C terminus of the Lsm4 variants, which were expressed in cells with genomically tagged Dcp2-GFP (>100 foci counted per condition). See Table S1.

Figure 6. Specific Interactions Can Synergize with Promiscuous Nonspecific Interactions to Drive Assembly

A) Cells expressing Dhh1-GFP, either genomically or as a plasmid-expressed Dhh1-GFP variant. Cells were deprived of glucose for 10 min to induced P-body assembly. B) Quantification of (A), depicting the percentage of cells containing at least one P-body. Error bars, ±SD. C) Cells expressing Dhh1 – LEA-SC or wild-type Dhh1. Cells were deprived of glucose for 10 min to induced P-body assembly, and visualized by genomically GFP-tagged Dcp2. D) Quantification of (C), percentage of cells with at least one P-body. *p < 0.05. Error bars, ±SD.

Figure 7. Model of RNP Granule Assembly and Contributions of IDRs

A) RNP granules assembly by a wide variety of specific and nonspecific interactions. B) A theoretical phase diagram depicting how the addition of nonspecific, IDR-driven interactions could decrease the critical concentration of assembly for higher-order structures.