Intrinsically disordered RGG/RG domains mediate degenerate specificity in RNA binding

Abstract

RGG/RG domains are the second most common RNA binding domain in the human genome, yet their RNA-binding properties remain poorly understood. Here, we report a detailed analysis of the RNA binding characteristics of intrinsically disordered RGG/RG domains from Fused in Sarcoma (FUS), FMRP and hnRNPU. For FUS, previous studies defined RNA binding as mediated by its well-folded domains; however, we show that RGG/RG domains are the primary mediators of binding. RGG/RG domains coupled to adjacent folded domains can achieve affinities approaching that of full-length FUS. Analysis of RGG/RG domains from FUS, FMRP and hnRNPU against a spectrum of contrasting RNAs reveals that each display degenerate binding specificity, while still displaying different degrees of preference for RNA.

Figure 1.

RRM and ZnF domains of FUS do not bind to the RNA with a high affinity. (A) Domain structure of FUS. (B) A trace amount of the DNMT RNA was incubated with increasing concentrations of FUS, RRM (267–373) or ZnF (422–453). Binding was analyzed by electrophoretic mobility shift assay (EMSA). b = bound DNMT RNA and f = free DNMT RNA. ‘(−)΄ shows no protein lane. (C) Binding curves of EMSA data. Error bars represent the S.D. of three independent titrations for each construct.

Figure 2.

Flanking RGG/RG domains impart the RNA binding activity of the RRM. (A) Representative EMSAs and (B) the corresponding binding curves showing binding of DNMT RNA to RGG1-RRM (165–373), RRM-RGG2 (267–422), RGG1-RRM-RGG2 (165–422) proteins. b = bound and f = free RNA. ‘(−)’ shows no protein lane. Error bars represent the S.D. of three independent titrations for each construct.

Figure 3.

Individual RGG/RG domains of FUS bind to RNA. (A) Representative EMSAs and (B) corresponding binding curves of individual MBP-RGG domains of FUS; RGG1 (165–267), RGG2 (372–422), RGG3 (454–501), in the presence of DNMT. b = bound and f = free RNA. ‘(−)’ shows no protein lane. Error bars represent the S.D. of three independent titrations for each construct.

Figure 4.

RGG/RG domains of FUS mediate high affinity binding to RNA. (A) Schematic illustration of RGG/RG domain mutations. Arginine amino acids of individual RGG/RG domains were converted to serine amino acids in SGG1, SGG2 and SGG3 mutants. In SGG4 mutant, arginine amino acids in all RGG/RG domains were converted to serines. (B) Representative EMSAs of mutant FUS proteins with the DNMT RNA and (C) corresponding binding curves. b = bound and f = free. ‘(−)’ shows no protein lane. Error bars represent the standard deviation of three independent titrations for each construct. (D) Western blot data of flag-tagged, wild-type and mutant FUS constructs expressed in HEK293T cells. (E) SDS-PAGE of radiolabeled RNA fragments cross-linked to flag-tagged FUS or SGG mutants of FUS. (F) Two technical replicates of three separate pull-downs were quantitated and average together. Error bars represent standard deviation.

Figure 5.

RGG/RG domains represent moderate preference for structured RNAs. Heat-map for affinity of RGG/RG domains and FUS constructs for each RNA. EMSA experiments were performed with DNMT and Sc1 RNAs containing G-quadruplexes, hRRD and mRRD RNAs with complex secondary structures, dsAU and dsGC simple double stranded RNAs, and GGUG, CRL and poly-A single stranded RNAs. K_D,app (μM) was represented with a color code for all combinations of RNA–protein with a data range from two or more independent experiments.

Figure 6.

Model for RNA recognition by RGG/RG domains. (A) RGG/RG proteins may bind RNA in a 1:1 interaction or as a member of a higher order complex. (B) Multiple RGG/RG domains as part of a larger protein complex may recognize long RNA sequences. (C) RGG/RG proteins have the flexibility to accommodate and bind tightly to a variety of complex RNA structures. (D) Higher order RGG/RG proteins may allow binding to multiple RNAs at the same time.