Crystal structures and RNA-binding properties of the RNA recognition motifs of heterogeneous nuclear ribonucleoprotein L: insights into its roles in alternative splicing regulation - PubMed (original) (raw)

Crystal structures and RNA-binding properties of the RNA recognition motifs of heterogeneous nuclear ribonucleoprotein L: insights into its roles in alternative splicing regulation

Wenjuan Zhang et al. J Biol Chem. 2013.

Abstract

Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is an abundant RNA-binding protein implicated in many bioprocesses, including pre-mRNA processing, mRNA export of intronless genes, internal ribosomal entry site-mediated translation, and chromatin modification. It contains four RNA recognition motifs (RRMs) that bind with CA repeats or CA-rich elements. In this study, surface plasmon resonance spectroscopy assays revealed that all four RRM domains contribute to RNA binding. Furthermore, we elucidated the crystal structures of hnRNP L RRM1 and RRM34 at 2.0 and 1.8 Å, respectively. These RRMs all adopt the typical β1α1β2β3α2β4 topology, except for an unusual fifth β-strand in RRM3. RRM3 and RRM4 interact intimately with each other mainly through helical surfaces, leading the two β-sheets to face opposite directions. Structure-based mutations and surface plasmon resonance assay results suggested that the β-sheets of RRM1 and RRM34 are accessible for RNA binding. FRET-based gel shift assays (FRET-EMSA) and steady-state FRET assays, together with cross-linking and dynamic light scattering assays, demonstrated that hnRNP L RRM34 facilitates RNA looping when binding to two appropriately separated binding sites within the same target pre-mRNA. EMSA and isothermal titration calorimetry binding studies with in vivo target RNA suggested that hnRNP L-mediated RNA looping may occur in vivo. Our study provides a mechanistic explanation for the dual functions of hnRNP L in alternative splicing regulation as an activator or repressor.

Keywords: Crystal Structure; Protein Structure; RNA Binding; RNA Looping; RNA Splicing; RNA-Protein Interaction; RNA-binding Proteins; RRM Domain; hnRNP-L.

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Figures

FIGURE 1.

RRM domains and domain organization of human hnRNP L. A, sequence comparison of RNP-2 and RNP-1 motifs in hnRNP L RRMs with the RRM family consensus. Nonconserved residues from the consensus are shaded gray. Conserved aromatic positions important for RNA binding are indicated by black triangles. B, schematic representation of the domain organization of hnRNP L and the constructs used in this study.

FIGURE 2.

Structure and RNA-binding surface mapping of hnRNP L RRM1. A, ribbon representation of hnRNP L RRM1 structure. ribonucleoprotein motifs are colored orange. Residues at key aromatic positions are color-coded by atom type (cyan, carbon; red, oxygen; blue, nitrogen). B, structure comparison of hnRNP L RRM1 (green) and PTB RRM1 in both RNA-free (blue, PDB code 1SJQ) and RNA-bound (red, PDB code 2AD9) states. RNA in the complex structure of PTB RRM1-CUCUCU is shown in magenta. Major structural differences are highlighted. The hydrogen bond between Thr-98 and Asn-172 in hnRNP L RRM1 is indicated. C, residues selected for mapping the RNA-binding surface of hnRNP L RRM1. Mutations of residues that reduced, increased, and had no effect on RNA binding are indicated in red, green, and yellow, respectively. D, sequence alignment of hnRNP L RRM1. Residues related to the spatial direction of the N-terminal loop (Thr-98 and Asn-172) are indicated by black triangles. Residues involved in RNA binding are designated with black stars. Different substitutions in hnRNP L RRM1 at the positions important for U2 and U4 recognition of PTB RRM1 are indicated by dots.

FIGURE 3.

Structure and RNA-binding surface mapping of hnRNP L RRM34. A, ribbon representation of hnRNP L RRM34 structure. Secondary structure elements of hnRNP L RRM34 are labeled. The structure is colored by domains as follows: RRM3 (residues 380–480), green; interdomain linker (residues 481–500), red; RRM4 (residues 501–588), blue. Disordered residues between β2 and β3 of RRM4 are modeled as dashed lines. B, direct interactions between RRM3 and RRM4. C, indirect interdomain interactions mediated by the linker. Residues involved in B and C are represented by sticks, and the dotted surfaces represent hydrophobic interaction surfaces. D, sequence alignment of human hnRNP L RRM34 with its homologues. Amino acids involved in the interdomain interactions are designated with black triangles below; residues involved in RNA binding are designated with black stars. Different substitutions in hnRNP L RRM34 at the positions important for U2 and U4 recognition of PTB RRM34 are indicated by dots. E and F, residues selected for mapping the RNA binding surface of hnRNP L RRM3 (E) and RRM4 (F). Residues whose mutations reduced and had no effect on RNA binding are indicated in red and yellow, respectively.

FIGURE 4.

Residues affecting the binding specificity of hnRNP L RRM34 in comparison with PTB RRM34. A, structure-based sequence alignment of hnRNP L RRM1 and PTB RRM1. Residues in PTB RRM1 involved in the recognition of C3 are indicated by triangles and those of U2 and U4 by stars, colored red for the hydrophobic interactions and black for the hydrogen bond interactions. Different substitutions in hnRNP L RRM1 at the positions important for U2 and U4 recognition of PTB RRM1 are indicated by dots. B, structure-based sequence alignment of hnRNP L RRM34 and PTB RRM34. Residues in PTB RRM34 involved in the special recognition of C3 are indicated by triangles and those of U2 and U4 by stars, colored red for the hydrophobic interactions and black for the hydrogen bond interactions. Different substitutions in hnRNP L RRM34 at the positions important for U2 and U4 recognition of PTB RRM34 are indicated by dots. C, comparison of the cavities of hnRNP L (cyan) with the corresponding binding cavities of PTB RRM1 (PDB code 2AD9) and RRM3 (PDB code 2ADC) (violet) for U4.

FIGURE 5.

FRET-based gel shift and steady-state FRET assays of hnRNP L RRM34 binding to three different RNAs. A, sequences of three different RNAs with the CA repeats underlined. B, schematic representation of the RNA looping mechanism of hnRNP L RRM34. C, FRET-based gel shift assays of the fluorophore-labeled RNAs in the absence and presence of hnRNP L RRM34. Donor emission in the figure is shown in green and acceptor emission in red. The figure was generated by superposition using Photoshop 11.0 (Adobe). D, EMSA for binding of hnRNP L RRM34 to U21, establishing a 1:1 stoichiometry of the complex. E, fluorescence emission spectra of labeled U21 in the absence (dashed line) and presence (solid line) of 750 n

hnRNP L RRM34. F, FRET efficiencies of labeled U5, U15, and U21 in the absence (black) and presence (gray) of 750 n

hnRNP L RRM34. G, FRET efficiency of U21 as a function of [hnRNP L RRM34]. The data were fitted with a modified Hill equation (see “Experimental Procedures”). Error bars were derived from three independent assays.

FIGURE 6.

hnRNP L RRM34 binds to U21 RNA in RNA-looped monomers but not dimers. A, schematic representation of the RNA-looped monomer. B, schematic representation of the dimer containing two hnRNP L RRM34 and two RNAs. C, cross-linking SDS-PAGE analysis of the compound form of hnRNP L RRM34 with RNA. The bands for protein·RNA complex are indicated. D, dynamic light scattering assays of hnRNP L RRM34 in the absence (black) and presence (red) of U21 RNA.

FIGURE 7.

Binding of hnRNP L RRM34 to its in vivo target RNA. A, electrophoretic mobility gel shift data for binding of 34-nt RNA to hnRNP L RRM34. The RNA sequence is shown above. Activation-responsive sequence motifs (underlined and in bold) of CD45 exon 5, which are separated by a 21-nt spacer, represent binding sites for individual hnRNP L RRM domains. B, ITC measuring binding of hnRNP L RRM34 to 34-nt RNA (top, raw titration data; bottom, integrated heat measurements). The curve was fitted using a single-site binding model with KD and n indicated.

FIGURE 8.

Models for hnRNP L regulation of alternative splicing. A, hnRNP L represses inclusion of an alternative exon in the case of the splicing regulation of exon 5 of CD45. hnRNP L binds two motifs flanking the ESE in exon 5 and changes the conformation of the ESE, consequently repressing its binding to the enhancer complex and blocking the activity of the splicing enhancer. B, hnRNP L promotes removal of a specific intron in the case of the splicing regulation of DAF. hnRNP L binds two distant CA clusters in intron 7, loops out the long in-between sequence, brings the splice sites into close proximity, and recruits U1 and U2 snRNP to recognize the 5′ and 3′ splice sites. Red boxes indicate hnRNP L-binding motifs.

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Crystal structures and RNA-binding properties of the RNA recognition motifs of heterogeneous nuclear ribonucleoprotein L: insights into its roles in alternative splicing regulation - PubMed (original) (raw)