The structure and selectivity of the SR protein SRSF2 RRM domain with RNA

Abstract

SRSF2 is a prototypical SR protein which plays important roles in the alternative splicing of pre-mRNA. It has been shown to be involved in regulatory pathways for maintaining genomic stability and play important roles in regulating key receptors in the heart. We report here the solution structure of the RNA recognition motifs (RRM) domain of free human SRSF2 (residues 9-101). Compared with other members of the SR protein family, SRSF2 structure has a longer L3 loop region. The conserved aromatic residue in the RNP2 motif is absent in SRSF2. Calorimetric titration shows that the RNA sequence 5'AGCAGAGUA3' binds SRSF2 with a K(d) of 61 ± 1 nM and a 1:1 stoichiometry. NMR and mutagenesis experiments reveal that for SFSF2, the canonical β1 and β3 interactions are themselves not sufficient for effective RNA binding; the additional loop L3 is crucial for RNA complex formation. A comparison is made between the structures of SRSF2-RNA complex with other known RNA complexes of SR proteins. We conclude that interactions involving the L3 loop, N- and C-termini of the RRM domain are collectively important for determining selectivity between the protein and RNA.

Figure 1.

(A) SRSF2 RRM sequence and secondary structure. (B–E) Structure of SRSF2 RRM: (B) ensemble structures. (C) cartoon representation and schematic colored blue to red N-terminus to C-terminus, loops, strands and helices labeled according to RRM consensus (1). (D) electrostatic surface (red −5, blue +5). (E) hydrophobic residue analysis of SRSF2 identified two surface exposed patches, one on the helical face (yellow) the other on the β-sheet face (cyan); the hydrophobic core of the molecule comprises residues from both helices (magenta) and strands (red), for clarity flexible C-terminus residues 94–101 are omitted.

Figure 2.

Alignment of the SR protein RRM domains. (A) PDB deposited structures colored according to overlay and identified by PDB number and name, all other RRMs colored grey and identified by uniprot number and name. Conserved residues for RNA binding are highlighted in yellow (B) Left: Overlay of backbone atoms of the molecular structures of all known SR RRM domains, backbone alignment using 57 residues (indicated in by dots beneath the sequence) with an RMSD of 1.18 Å. Structures shown of human SR family RRM domains; SRSF1 (1X4A–RSGI), SRSF2B (2DNM–RSGI), SRSF7 [2HVZ (16)] and SRSF3 [2I2Y, 2I38 (16)]. Right: Cartoon representation of structures; for clarity only two SR–RRM domains are shown; SRSF2 and SRSF3, the SRSF3 structure used for this alignment is from PDB ID 2I2Y, the only SR–RRM structure determined in the presence of RNA.

Figure 3.

Histogram of chemical shift changes—residues with combined shifts greater than 0.15 ppm (orange) and 0.25 ppm (yellow) marked on structure inset. Filled circle represents NH peak not assigned, open circles represent peaks that broaden or cannot be tracked upon titration and asterisks represent overlapping NH peaks. Significant shift changes/line-broadening can be seen for residues in the β-strands as well and residues in L3, in particular residues Y50 and T51, and the N-termini.

Figure 4.

SRSF2:RNA UV cross-linking. Various purified proteins (BSA, GB1-6His-SRSF2 9-101 wild-type and point mutants) were incubated with ³²P-labelled 9-mer RNA (AGCAGAGUA) before binding reactions were irradiated with UV (+) or not (−) and analysed by 15% SDS–PAGE stained with Coomassie and autoradiography.

Figure 5.

Isothermal titration calorimetry curves for WT and K52A mutant fit to a one-site model. (A) WT SRSF2 with AGCAGAGUA (25 mM PO₄^3-, 25 mM KCl, 25°C) curve fitting to a one-site 1:1 model yields fit parameters: N (stoichiometry ratio) = 1.03, K_d = 6.17 × 10⁻⁸M, ΔH = −21.3 kcal/mol and ΔS = −38.4 cal mol⁻¹K⁻¹. (B) K52A SRSF2 with AGCAGAGUA (25 mM PO₄^3-, 25 mM KCl, 25°C), curve fitting to a one-site model yields fit parameters: N = 0.953, K_d = 1.63 × 10⁻⁷M, ΔH = −30 kcal/mol and ΔS = −69.5 cal mol⁻¹K⁻¹.

Figure 6.

(A) NOEs used to derive the model of SRSF2–RRM bound to 9-mer AGCAGAGUA RNA; V10-Ade6, D48-Gua2, Y50-Gua2 and Y92-Uri8. (B) Left: Ensemble of 10 structures from CNS calculations that contribute to the lowest energy cluster. (C) Left: Ensemble of five structures from CNS calculations that contribute to the second cluster. In both (B) and (C) the mobility of loop 3 and terminal regions afford a great degree of freedom to the orientation of the RNA. Right: representative structure (closest to mean) from each cluster with side chain residues shown for the incorporated intermolecular NOEs. In addition conserved hydrophobic residues F57 and F59 (pale yellow) are found to be involved in the binding.