Molecular basis for the specific and multivariant recognitions of RNA substrates by human hnRNP A2/B1

Abstract

Human hnRNP A2/B1 is an RNA-binding protein that plays important roles in many biological processes, including maturation, transport, and metabolism of mRNA, and gene regulation of long noncoding RNAs. hnRNP A2/B1 was reported to control the microRNAs sorting to exosomes and promote primary microRNA processing as a potential m⁶A "reader." hnRNP A2/B1 contains two RNA recognition motifs that provide sequence-specific recognition of RNA substrates. Here, we determine crystal structures of tandem RRM domains of hnRNP A2/B1 in complex with various RNA substrates, elucidating specific recognitions of AGG and UAG motifs by RRM1 and RRM2 domains, respectively. Further structural and biochemical results demonstrate multivariant binding modes for sequence-diversified RNA substrates, supporting a RNA matchmaker mechanism in hnRNP A2/B1 function. Moreover, our studies in combination with bioinformatic analysis suggest that hnRNP A2/B1 may mediate effects of m⁶A through a "m⁶A switch" mechanism, instead of acting as a direct "reader" of m⁶A modification.

Fig. 1

Overview of the structure and ITC of hnRNP A2/B1 in complex with 8mer RNA. a Schematic representation of the domain architecture of hnRNP A2/B1. RRM: RNA recognition motif, PrLD: prion-like domain, NLS: nuclear location signal, RGG: arginine-glycine-glycine box. b ITC results of hnRNP A2/B1(12–195) with 8mer and 10mer RNA targets. Solid lines indicate nonlinear least-squares fit the titration curve, with ΔH (binding enthalpy kcal mol⁻¹), Ka (association constant), and N (number of binding sites per monomer) as variable parameters. Calculation values for K_d (dissociation constant) are indicated. c Cartoon representation of RRMs in complex with 8mer RNA. The RNA backbone is colored in yellow shown by stick. The RRMs are colored in purple-blue. d Molecules from two adjacent asymmetric units. The molecule from another asymmetric unit is colored in green. e Intermolecular contacts between RRM1 and 8mer RNA from A₁ to G₃. All dashed lines in this study indicate distance <3.2 Å. f Schematic showing RRMs interactions with 8mer RNA 5′-AGGACUGC-3′. g Close-up view showing the specific recognition of A₁, G₂, G₃, A₄, and U₆

Fig. 2

Overview of RRMs in complex with 10mer RNA. a Cartoon representation of RRMs in complex with 10mer RNA 5′-AAGGACUAGC-3′. The RNA backbone is colored in yellow shown by stick. The molecule from the adjacent asymmetric unit is colored in green. b Surface representation of RRMs–10mer complex. c Intermolecular contacts between RRM1 and residues of 10mer RNA 5′-AAGG-3′, and RRM2 with residues of RNA 5′-ACUAGC-3′. d Superposition of 8mer and 10mer RNA substrates. The 8mer RNA 5′-AGGACUGC-3′ is colored in marine and the 10mer RNA 5′-AAGGACUAGC-3′ is colored in yellow

Fig. 3

Detailed interactions between RRMs and 10mer RNA. a Schematic showing RRMs interactions with 10mer RNA sequence. b Close-up view showing the specific recognition from A₁ to G₈. c Mutagenesis study by ITC experiments between protein mutants and 10mer RNA substrate. d ITC experiments between hnRNP A2/B1 RRMs and RNA mutants A1G, G2C, G3C, U6G, and A7U

Fig. 4

RNA mutants indicate mutivariant-binding mode of hnRNP A2/B1 RRMs. a Structure of RRMs (12–195) in complex with A1G-RNA 5′-AGGGACUAGC-3′. b Structure of RRMs (12–195) in complex with U6G RNA 5′-AAGGACGAGC-3′. c Structure of RRMs (12–195) in complex with A7U-RNA 5′-AAGGACUUGC-3′. d Intermolecular contacts between RNA and RRMs in A1G complex. RRM1 is colored in purple-blue, RRM2 is colored in green. e Schematic representation of the comparison of different intermolecular interactions between 10mer RNA 5′-AAGGACUAGC-3′ and A1G-RNA 5′-AGGGACUAGC-3′. f Close-up view showing the specific recognition from A₁ to G₃. g Intermolecular contacts between RNA and RRMs in U6G complex. h Schematic representation of intermolecular interactions in the U6G complex. i Close-up view showing the specific recognition of A₁, G₆, and A₇ in U6G complex. j Intermolecular contacts between RNA and RRMs in A7U complex. k Schematic representation of intermolecular interactions in A7U complex. l Close-up view showing the specific recognition of U₆, U₇, and the stacking interactions involved in A₄, U₆, F157, and M193 in A7U complex

Fig. 5

The RNA binding by RRMs adopts an antiparallel mode. a Superimposition of different structure complex. 8mer-RNA: 5′-AGGACUGC-3′ is colored in orange, 10mer-RNA: 5′-AAGGACUAGC-3′ is colored in red, A1G-RNA: 5′-AGGGACUAGC-3′ is colored in green, U6G: RNA 5′-AAGGACGAGC-3′ and A7U-RNA: 5′-AAGGACUUGC-3′ are colored in cyan and purple, respectively. b Interactions between RRM1 and RRM2; the amino acids participating in interactions are colored in green and RNA is colored in yellow. c The overall structure of hnRNP A2/B1-10mer RNA complex. d hnRNP A1 in complex with DNA. e–h The overall structure of HuD-RNA, HuR-RNA, PABP-RNA, and TDP-43-RNA. From c to h, RRM1 is colored in blue and RRM2 is colored in red, the linker is colored in green pointed out with a black arrow, the RNA backbone is colored in yellow shown by stick. i The overall structure of PTB–RNA complex with an antiparallel RNA-binding mode. RRM3 is colored in blue and RRM4 is colored in red, other labels are the same as from c to h. j–l Crystal packing interactions in 10mer, A1G and U6G. To illustrate the detailed packing interactions of hnRNP A2/B1 carrying two antiparallel RNA stands with other hnRNP A2/B1 molecules, three hnRNP A2/B1 molecules and two RNA strands of each complex are selected to show

Fig. 6

hnRNP A2/B1 does not specifically recognize m⁶A-modified RNA. a Surface representation of the environment around A₄ in 8mer RNA complex. b Surface representation of the environment around A₄ in 10mer RNA complex. c Surface representation of the canonical N⁶-methylated adenosine binding mode in HsYTHDC1. The aromatic cage is circled with yellow dashline. d ITC data of hnRNP A2/B1(12–195) with 8mer and 10mer RNA targets carried N⁶-methylated adenosine. e EMSA experiment shows the binding affinity of full-length hnRNP A2/B1 with 5′-FAM-labeled RNA substrates with or without m⁶A modification. Uncropped gel image is shown in Supplementary Fig. 7e. The data represent the mean of three independent experiments, with standard deviation (SD) values indicated by error bars. f YTHDC1 shows preferential binding to m⁶A sites in nuclear RNA compared to hnRNP A2/B1. For hnRNP A2/B1, the m⁶A-Seq reads that overlapped with each m⁶A site was plotted on the x-axis, and the HITS-CLIP reads that overlap with each site were plotted on the y-axis. A similar analysis was used to examine YTHDC1 binding at these m⁶A sites. miCLIP reads that overlapped with the m⁶A sites were plotted on the x-axis, and the YTHDC1 iCLIP reads that overlapped with the m⁶A sites were plotted on the y-axis. g YTHDC1-m⁶A tag cluster overlap. A Venn diagram indicating the cluster overlap is shown. Roughly, 43 and 56% of miCLIP tag clusters from total cellular RNA and poly(A) RNA showed a significant overlap with the YTHDC1 iCLIP clusters, respectively