NMR structure of the three quasi RNA recognition motifs (qRRMs) of human hnRNP F and interaction studies with Bcl-x G-tract RNA: a novel mode of RNA recognition

Abstract

The heterogeneous nuclear ribonucleoprotein (hnRNP) F belongs to the hnRNP H family involved in the regulation of alternative splicing and polyadenylation and specifically recognizes poly(G) sequences (G-tracts). In particular, hnRNP F binds a G-tract of the Bcl-x RNA and regulates its alternative splicing, leading to two isoforms, Bcl-x(S) and Bcl-x(L), with antagonist functions. In order to gain insight into G-tract recognition by hnRNP H members, we initiated an NMR study of human hnRNP F. We present the solution structure of the three quasi RNA recognition motifs (qRRMs) of hnRNP F and identify the residues that are important for the interaction with the Bcl-x RNA by NMR chemical shift perturbation and mutagenesis experiments. The three qRRMs exhibit the canonical betaalphabetabetaalphabeta RRM fold but additional secondary structure elements are present in the two N-terminal qRRMs of hnRNP F. We show that qRRM1 and qRRM2 but not qRRM3 are responsible for G-tract recognition and that the residues of qRRM1 and qRRM2 involved in G-tract interaction are not on the beta-sheet surface as observed for the classical RRM but are part of a short beta-hairpin and two adjacent loops. These regions define a novel interaction surface for RNA recognition by RRMs.

Figure 1

Overview of the structures of qRRM1, qRRM2 and qRRM3 of human hnRNP F. (A) Sequence alignment of the three qRRMs of hnRNP F. Identical residues are colored red and homologous residues are colored blue. Residues corresponding to RNP1 and RNP2 sequences are boxed. (B) Comparison of the consensus RNP1 and RNP2 sequence of RRMs with corresponding residues of hnRNP F qRRMs. Consensus residues important for RNA binding are colored red. (C) Overlay of the 20 final structures and ribbon representation of the lowest energy structure of qRRM1, qRRM2 and qRRM3. Figures were generated with MOLMOL (51).

Figure 2

The hydrophobic cluster formed between the C-terminal α-helix and the β-sheet of qRRM1 and qRRM2. In qRRM1, M93, V96 and L97 of the C-terminal α-helix interact with V12, H44, I46 and F58 of the β-sheet. In qRRM2, V191 of the C-terminal α-helix interact with F112, T142 and F156 of the β-sheet. Figures were generated with MOLMOL (51).

Figure 3

Relaxation studies of hnRNP F qRRM1–2. (A) HSQC spectrum of qRRM1–2 (black) overlaid with HSQC spectra of qRRM1 (green) and qRRM2 (red). (B) Heteronuclear NOE values (top panel), T1 relaxation rates (middle panel) and T2 relaxation rates from CPMG experiments (bottom panel). The secondary structure elements are also displayed.

Figure 4

MR chemical shift perturbation experiments of qRRM1–2 and qRRM3 with Bcl-x G-tract RNAs. (A) HSQC of free qRRM1–2 (black) overlaid with the HSQC of qRRM1–2 in complex with the Bcl-x G-tract RNA, CGGGAUGGGGUA, in a 1:1 ratio (red). Peaks corresponding to residues showing large chemical shift changes upon RNA binding are labeled and the shifts are indicated. Boxed peaks correspond to peaks for which no assignment could be derived in the bound form. (B) HSQC of free qRRM3 (black) overlaid with the HSQC of qRRM3 in complex with CUGGGGU in a 1:1 ratio (red). Boxed peaks tend to disappear during RNA titration. (C) Combined chemical shift perturbations (Δδ=[(δ_HN)² + (δ_N/6.51)²]^1/2) of qRRM1–2 upon binding with Bcl-x G-tract RNA as a function of qRRM1–2 amino acid sequence. Red bars correspond to residues for which no assignments could be derived in complex with RNA. (D) Sequence alignment of qRRM1, qRRM2 and qRRM3 of human hnRNP F. Residues showing a significant chemical shift perturbation (>0.1) or that disappear upon RNA binding are colored red. Residues corresponding to RNP1 and RNP2 sequences are boxed.

Figure 5

Residues of qRRM1 and qRRM2 showing a large chemical shift perturbation are clustered in the β-hairpin, the β1−α1 loop and the β2−β3 loop. (A) Ribbon representation of qRRM1 and qRRM2. Aromatic and positively charged side chains showing a significant chemical shift perturbation are displayed and labeled. Figures were generated with MOLMOL (51). (B) Surface representation of qRRM1 and qRRM2 colored according to electrostatic potential (red and blue indicate negative and positive charges, respectively). Figures were generated with PYMOL ().