Distinct roles of hnRNPH1 low-complexity domains in splicing and transcription

Abstract

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins that control key events in RNA biogenesis under both normal and diseased cellular conditions. The low-complexity (LC) domain of hnRNPs can become liquid-like droplets or reversible amyloid-like polymers by phase separation. Yet, whether phase separation of the LC domains contributes to physiological functions of hnRNPs remains unclear. hnRNPH1 contains two LC domains, LC1 and LC2. Here, we show that reversible phase separation of the LC1 domain is critical for both interaction with different kinds of RNA-binding proteins and control of the alternative-splicing activity of hnRNPH1. Interestingly, although not required for phase separation, the LC2 domain contributes to the robust transcriptional activation of hnRNPH1 when fused to the DNA-binding domain, as found recently in acute lymphoblastic leukemia. Our data suggest that the ability of the LC1 domain to phase-separate into reversible polymers or liquid-like droplets is essential for function of hnRNPH1 as an alternative RNA-splicing regulator, whereas the LC2 domain may contribute to the aberrant transcriptional activity responsible for cancer transformation.

Keywords: hnRNPH1; low-complexity domain; phase separation; splicing; transcription.

Fig. 1.

In vitro phase separation of hnRNPH1. (A) Hydrogel droplets composed of mCherry-linked full-length (amino acids 2 through 449, Top Left), C-terminal half (C-term, amino acids 192 through 449, Top Middle), or LC1 domain (amino acids 192 through 280, Top Right) of hnRNPH1 were imaged by confocal microscopy (CM) (Materials and Methods). Polymers of each hydrogel droplet sample shown in Bottom panels were visualized by TEM (Materials and Methods). (Scale bar, 0.1 µm) (B) The polymer samples of mCherry-linked ySup35 or hnRNPH1 C terminus were incubated with indicated levels of SDS. The samples were then migrated through the agarose gel containing SDS, and the polymers or monomers were visualized by Western blotting using anti-mCherry antibodies.

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 ΔLC1 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.

The LC1 domain of hnRNPH1 is required for interactions with different kinds of RNA regulatory proteins. (A) Different regions of hnRNPH1 in the form of GFP-linked protein at equal molar concentrations were incubated with mCherry hydrogel droplets composed of LC domains of FUS, TAF15, DHX9, hnRNPA1, hnRNPA2, and hnRNPF. The intensity of the hydrogel binding was analyzed using confocal microscopy. (B) HEK-293T cells were transfected with Flag-tagged WT, ΔLC1, or ΔLC2 hnRNPH1 constructs. Immunoprecipitation was performed using anti-Flag antibodies, and coprecipitated proteins were analyzed by Western blotting using antibodies for indicated proteins. Representative images are shown from more than three independent immunoprecipitation experiments.

Fig. 4.

Effects of Y-to-S mutations on phase separation and function of hnRNPH1. (A) The recombinant proteins of the WT or Y/F-to-S mutant hnRNPH1 LC1 domains with N-terminal GFP tag were incubated with hydrogel droplets composed of the mCherry-linked hnRNPH1 C-terminal half. Upon overnight incubation, hydrogel-trapping of the GFP-linked LC1 domains was analyzed using confocal microscopy. (B) For LLD formation assay, 1, 3, or 10 µM of the purified recombinant proteins of GFP-linked WT, Y210/219S (Y2S), and Y236/240/243S (Y3S) were incubated in buffer containing 150 mM NaCl and 10% level of the crowding agent, PEG. The LLDs were visualized by light microscopy. (Scale bar, 10 µm.) (C) GFP-linked proteins of WT, Y2S, or Y3S LC1 domain were incubated with different kinds of mCherry hydrogel droplets. Hydrogel binding of the GFP-linked proteins was analyzed by confocal microscopy. (D) RT-PCR was performed to analyze the splicing of the exon 5 of C2orf18, exon 5 of CDK2, exon 11 of Man2a2, and exon 11 of PEX26 transcripts. The mean value of PSI levels obtained from three independent experiments is shown.

Fig. 5.

The LC2 domain of hnRNPH1 is required for transcriptional activity as a fusion protein with DBD. (A) The indicated regions of hnRNPH1 were linked to the DBD of GAL4 and assayed for activation of GAL4-dependent firefly luciferase reporter gene in HEK-293T cells. Relative luciferase activity compared to GAL4-DBD-only control. (B) Transcriptional activity of the WT or Y-to-S mutant LC2 domains of hnRNPH1 linked to GAL4-DBD were analyzed in HEK-293T cells. (A and B) One-way ANOVA was used to evaluate statistical significance. Mean ± SEM from three independent assays; ns, not significant versus control; *P < 0.01, **P < 0.001, and ***P < 0.0001 versus control; and ^#P < 0.01, ^##P < 0.001, and ^###P < 0.0001 versus WT. (C) Melting of mCherry hydrogel droplets composed of WT or Y408S C-terminal half of hnRNPH1 15% levels of an aliphatic alcohol, 1,6-HD. The hydrogel droplets were imaged by confocal microscopy at indicated time points after addition of 1,6-HD. (D) Different kinds of mCherry hydrogel droplets were challenged with GFP-linked proteins of WT or Y408S LC2 domains of hnRNPH1. Trapping of the GFP-linked proteins by the hydrogel droplets was analyzed by confocal microscopy.