Fig 2: Hydrogel binding and phase separation of the LC1 domain of hnRNPH1. (A) Hydrogel droplets composed of mCherry-linked C-terminal half of hnRNPH1 were challenged with soluble proteins of GFP-linked full-length (FL), N-terminal half (N-term), C-terminal half (C-term), LC1 domain, LC2 domain, or ?LC1 hnRNPH1. Upon overnight incubation, trapping of the GFP-linked proteins by the mCherry hydrogel droplets was detected using confocal microscopy. (B) Phase separation of GFP-linked different regions of hnRNPH1 proteins into LLDs. Different concentrations (3 or 10 µM) of GFP-linked recombinant proteins were incubated in the presence of 10% PEG for indicated time periods. The LLDs were visualized using light microscopy. (Scale bar, 10 µm.) (C) Melting of LLDs formed from the hnRNPH1 LC1 domain by 1,6- or 2,5-HDs. The droplets were visualized using light microscopy. (Scale bar, 10 µm.) (D) Incorporation of 1 µM of mCherry alone, mCherry-linked LC1, RRM3, or LC2 domains of hnRNPH1 into LLDs composed of 10 µM GFP-linked LC1 domain of hnRNPH1. Upon overnight incubation, the mCherry signals incorporated to the GFP LLDs were assessed using confocal microscopy. (Scale bar, 20 µm.)

Fig 3: A subgroup of RNA G-quadruplex regions is enriched near HNRNPH1 binding sites in the human genome. (A) Coverage plots (left) and read density heat maps (right) represent binding read count for the indicated hnRNPs near RNA G-quadruplex sites (±1 Kb). The RNA targets and binding sites for each hnRNP were obtained from iCLIP or eCLIP data sets (Uren et al. 2016; Van Nostrand et al. 2016). The RNA G-quadruplex sites were obtained from the sequencing data set GSE77282 (Kwok et al. 2016). (B) Overrepresented sequence motifs at HNRNPH1 binding sites that overlap with either the highest-ranked 1500 RNA guanine quadruplex regions (binned into sets of 500) or all exonic regions containing potential RNA guanine quadruplexes.

Fig 4: Specificity of the HNRNPH1 and rG1 oligomer interaction. (A) TC32 cells were transfected with siRNA or synthetic ssRNA oligomer for 24 h and followed by streptavidin pull-down. Interaction between HNRNPH1 and rG1/rG1mt1 was confirmed by immunoblotting for HNRNPH1. (UTR) Untreated cells. (B) Chemiluminescent EMSA gel for 3'-biotinylated EWSR1 exon 8 rG1 and rG1mt1 at varying concentrations of purified HNRNPH1. Wedges indicate decreasing amounts of purified HNRNPH1 (20, 10, 5, 2.5, and 1.25 nM). Arrowhead indicates mobility shift for each labeled probe. (C) The fold-enrichment for HNRNPH1-bound rG1 at varying concentrations of nonbiotinylated rG1 determined by comparison of chemiluminescent signals from pull-down performed using HNRNPH1 antibody and an IgG isotype control. Data are shown as the mean ± SEM of three technical replicates. (D) A schematic representation of the protein domains of HNRNPH1 and a panel of recombinant His-tagged HNRNPH1 domains used in this study. All domain annotations are adapted from Van Dusen et al. (2010). Schematic diagrams were constructed using Domain Graph (DOG), version 2.0 (Ren et al. 2009). (E) The binding of full-length HNRNPH1 protein to a rG1 RNA target surface by SPR is shown. Black lines represent the binding responses for HNRNPH1 at the indicated concentrations on each sensorgram. HNRNPH1 was exposed to the surface for 2 min (association phase) followed by 10 min flow of running buffer (dissociation phase). Data were fit globally to a 1:1 binding model and the respective fits are shown in red. The resulting parameter values are provided in Table 1. (F) The binding of full-length HNRNPH1 protein to a rG1mt1 RNA target surface by SPR is shown. Black lines represent the binding responses for HNRNPH1 at 15.6, 31.3, 62.5, and 125 nM. HNRNPH1 was exposed to the surface as described in E; however, no binding was observed.

Fig 5: HNRNPH1-mediated processing of EWSR1 exon 8 containing pre-mRNAs. (A) qPCR assessment of EWS–FLI1, EWSR1, HNRNPH1, HNRNPH2, and HNRNPF mRNA expression in HNRNPH1, HNRNPH2, or HNRNPF silenced TC32 EWS cells (48 h). Data are normalized to the geomean of reference genes, ACTB, RPL27, and NACA, and expressed relative to siNeg-transfected cells (mean ± SEM, n = 3). (**) P < 0.01; (***) P < 0.001 compared to siNeg-transfected cells. For additional experimental details, refer to Supplemental Table S7. (B) Expression of selected EWSR1 and EWS–FLI1 exon–exon junctions from targeted RNA-seq data analyzed using splicing junction analysis for HNRNPH1-silenced HEK-293T cells (top graph) and TC32 EWS cells (bottom graph). Data are expressed relative to siNeg-transfected cells (48 h, mean ± SEM, n = 3). (C) Schematic representation of putative HNRNPH1-dependent regulation of EWSR1 exon 8 containing pre-mRNAs.