Intrinsically disordered regions and RNA binding domains contribute to protein enrichment in biomolecular condensates in Xenopus oocytes

Abstract

Proteins containing both intrinsically disordered regions (IDRs) and RNA binding domains (RBDs) can phase separate in vitro, forming bodies similar to cellular biomolecular condensates. However, how IDR and RBD domains contribute to in vivo recruitment of proteins to biomolecular condensates remains poorly understood. Here, we analyzed the roles of IDRs and RBDs in L-bodies, biomolecular condensates present in Xenopus oocytes. We show that a cytoplasmic isoform of hnRNPAB, which contains two RBDs and an IDR, is highly enriched in L-bodies. While both of these domains contribute to hnRNPAB self-association and phase separation in vitro and mediate enrichment into L-bodies in oocytes, neither the RBDs nor the IDR replicate the localization of full-length hnRNPAB. Our results suggest a model where the additive effects of the IDR and RBDs regulate hnRNPAB partitioning into L-bodies. This model likely has widespread applications as proteins containing RBD and IDR domains are common biomolecular condensate residents.

Figure 1:. hnRNPAB X2 is a novel cytoplasmic splice isoform of hnRNPAB that is enriched in L-bodies.

(a) Stage II oocytes were microinjected with Cy5-labeled LE RNA (magenta, a′) and immunostained for endogenous hnRNPAB (green, a) using antibodies raised against Xenopus hnRNPAB. The overlap is shown in a″. (b) Cy5-labeled LE RNA (magenta, b′) was microinjected into stage II oocytes expressing mCherry-tagged canonical hnRNPAB, as detected by immunostaining with anti-mCherry (green, b). The overlap is shown in b″. (c) Schematics of canonical hnRNPAB, hnRNPAB X2 and hnRNPABΔPY. C-terminal sequence is shown below each, with the PY NLS indicated in red, the protein coding sequence (CDS) in gray, and the 3′UTR shown in orange. (d) RNA isolated from stage II oocytes was used to measure the relative expression of hnRNPAB X2 compared to canonical hnRNPAB by qPCR. ΔCt values were calculated normalizing to refence gene vg1 (* indicates p<0.05 by T-test). Error bars represent standard deviation from the mean, n=3. (e) Cy5-labeled LE RNA (magenta, e′) was microinjected into stage II oocytes expressing mCherry-tagged hnRNPAB X2, as detected by immunostaining with anti-mCherry (green, e). The overlap is shown in e″. (f) Cy5-labeled LE RNA (magenta, e′) was microinjected into stage II oocytes expressing mCherry-tagged hnRNPABΔPY, as detected by immunostaining with anti-mCherry (green, e). The overlap is shown in e″. Confocal sections (a-b, e-f) are shown with the vegetal hemisphere at the bottom; scale bars=100μm.

Figure 2:. hnRNPAB X2 localizes to L-bodies through its RBD and IDR.

(a) Schematics of domain constructs. (b-e) Stage II oocytes expressing (b) mCh-hnRNPAB X2, (c) mCh-RBD, (d) mCh-IDR, or (e) free mCh (green; detected by anti-mCherry IF) were co-microinjected with Cy5 LE RNA (magenta, b′-e′) to label L-bodies. Colocalization (white) is shown in the merged confocal images (b′-e′). Scale bars=100μm. (f) Scoring of L-body enrichment (n=21 oocytes) where 0 indicates no enrichment of the protein in L-bodies (as marked by LE RNA), 1 indicates modest enrichment, 2 indicates moderate enrichment and 3 indicates strong enrichment. Shown are levels of L-body enrichment for hnRNPAB X2 (green), RBD (blue), IDR (orange), and mCherry (red). n=21 oocytes, error bars represent standard error of the mean, **** indicates p <0.001and ns indicates p >0.05. Statistics shown are an Ordinary one-way ANOVA followed by Tukey’s multiple comparisons.

Figure 3:. hnRNPAB X2 and its domains self-assemble and phase separate in vitro.

DIC micrographs of 50 μM MBP-fusions of (a) full-length hnRNPA2 X2, (b) IDR, (c) RBD, or (d) control (free MBP) proteins in 20 mM NaPi (pH 7.4), 150 mM NaCl, and 10% PEG. Irregularly shaped assemblies are observed for hnRNPA2 X2 full-length (a), while round droplets consistent with liquid-liquid phase separation are observed for hnRNPAB X2 IDR and RBD (b-c) domains. No phase separation is observed for MBP alone (control, d). Images are representative from two biological replicates (with independently expressed and purified protein). Scale bars=20 μm.

Figure 4:. hnRNPAB X2 dynamically associates with L-bodies via its RBD and IDR.

(a) Shown is an image of the vegetal cytoplasm of a stage II oocyte microinjected with mCh-hnRNPAB X2. FRAP was conducted such that an individual L-body was partially bleached (a′), to allow for recovery (a″) both from within the L-body and from its environment. The 10μm² ROI is indicated by a white box; scale bar=10μm. (b) Stage II oocytes were microinjected with mCherry (mCh), mCh-hnRNPAB X2, mCh-RBD or mCh-IDR RNA to express the mCh-tagged proteins, along with Cy5 LE RNA to mark L-bodies. Normalized FRAP recovery curves are shown (n=21 oocytes per fusion protein); error bars represent SEM. Measurements were taken at 5 second intervals over 100 iterations. (c) Plateau values (% mobile fraction) for each construct are shown (ns indicates not significant, * indicates p<0.05). Error bars represent SEM. Statistics shown are an Ordinary one-way ANOVA with Tukey’s multiple comparisons. (d) T_1/2 measurements are shown for each construct, measured as the average time at which the construct recovers half of its plateau value (ns indicates not significant, *** indicates p<0.001, **** indicates p<0.0001). Error bars represent SEM. Statistics shown are an Ordinary one-way ANOVA with Tukey’s multiple comparisons.

Figure 5:. The hnRNPAB X2 IDR acts to stabilize proteins in L-bodies.

(a) The hnRNPAB X2 IDR was fused to the C-terminus of PTBP3 (PTBP3+X2IDR) and tagged with mCherry (mCh). (b) Stage II oocytes were microinjected with mCh-PTBP3 RNA to express the encoded protein (green, detected by anti-mCh IF) along with Cy5 LE RNA to label L-bodies (magenta, b′). The merge is shown in b″; scale bar=100μm. (c) Stage II oocytes were microinjected with mCh-PTBP3+IDR to express the encoded protein (green, detected by anti-mCh IF) along with Cy5 LE RNA (magenta, c′) to label L-bodies. The merge is shown in c″; scale bar=100μm. (d) Stage II oocytes were microinjected with RNA encoding PTBP3 or PTBP3+X2IDR, along with Cy5 LE RNA to label L-bodies. Normalized FRAP recovery curves are shown (n=21 oocytes); error bars represent SEM. Measurements were taken at 5 second intervals over 100 iterations. (e) Average percent mobile fractions for the two constructs are shown. Error bars represent SEM; ** indicates p<0.01, ns indicates not significant (p>0.05). Statistics shown are an Ordinary one-way ANOVA with Tukey’s multiple comparisons.

Figure 6:

A model for how RBDs and IDRs additively determine a protein’s localization (hnRNPAB) to and dynamics within L-bodies. (a) While RNA (magenta) is highly stable within L-bodies (grey), L-body associated proteins (blue, gold) dynamically associate with the RNA and other proteins. The strength of these associations determines the degree of their restriction to L-bodies: proteins that interact strongly with L-bodies (blue) show a greater degree of enrichment to L-bodies and lower degrees of dynamic activity, while proteins that interact more weakly (gold) are less enriched and more dynamic. (b-c) The association of a protein with L-bodies can be tuned by modifying its interaction domains. Additional interaction domains (b) may stabilize a protein within L-bodies, while removal of interaction domains (c) may serve to destabilize a protein within L-bodies. This principle holds true for both RBDs (represented by boxes) and IDRs (represented by lines).