Structural basis for m3G-cap-mediated nuclear import of spliceosomal UsnRNPs by snurportin1 - PubMed (original) (raw)

Structural basis for m3G-cap-mediated nuclear import of spliceosomal UsnRNPs by snurportin1

Anja Strasser et al. EMBO J. 2005.

Abstract

In higher eukaryotes the biogenesis of spliceosomal UsnRNPs involves a nucleocytoplasmic shuttling cycle. After the m7G-cap-dependent export of the snRNAs U1, U2, U4 and U5 to the cytoplasm, each of these snRNAs associates with seven Sm proteins. Subsequently, the m7G-cap is hypermethylated to the 2,2,7-trimethylguanosine (m3G)-cap. The import adaptor snurportin1 recognises the m3G-cap and facilitates the nuclear import of the UsnRNPs by binding to importin-beta. Here we report the crystal structure of the m3G-cap-binding domain of snurportin1 with bound m3GpppG at 2.4 A resolution, revealing a structural similarity to the mRNA-guanyly-transferase. Snurportin1 binds both the hypermethylated cap and the first nucleotide of the RNA in a stacked conformation. This binding mode differs significantly from that of the m7G-cap-binding proteins Cap-binding protein 20 (CBP20), eukaryotic initiation factor 4E (eIF4E) and viral protein 39 (VP39). The specificity of the m3G-cap recognition by snurportin1 was evaluated by fluorescence spectroscopy, demonstrating the importance of a highly solvent exposed tryptophan for the discrimination of m7G-capped RNAs. The critical role of this tryptophan and as well of a tryptophan continuing the RNA base stack was confirmed by nuclear import assays and cap-binding activity tests using several snurportin1 mutants.

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Figures

Figure 1

Structure of human snurportin1 (residues 97–300) with bound m3GpppG-cap dinucleotide (PDB accession code 1XK5). Ribbon plot of the m3G-cap-binding domain with β-sheets coloured in blue, α-helices in green and loop regions in grey. Secondary structure motifs are numbered consecutively from the N-terminus to the C-terminus. The m3GpppG dinucleotide is depicted in ball-and-stick mode with nitrogen atoms in blue, carbons in grey, oxygens in red and phosphorus atoms in green.

Figure 2

The m3G-cap-binding pocket. Snurportin1 is shown in ribbon presentation and coloured in green. Side chains of residues interacting with the m3G-cap dinucleotide are shown in ball-and-stick mode. The m3G-cap dinucleotide is coloured as described in Figure 1. (A) Stereo view of the cap-binding pocket. Side chains forming hydrogen bonds or hydrophobic contacts with the cap are coloured as the m3G-cap, and water molecules involved in the interaction are shown as yellow balls. Hydrogen bonds are depicted as dashed grey lines. (B) Base stack of the m3GpppG dinucleotide, whereas Trp276 and Trp107 are coloured yellow. (C) Distances between both bases of the cap dinucleotide, between the m3G-base and Trp276 and between the atoms of the dimethylamine of the cap base and Trp107 are depicted as dashed black lines. Numbers indicate the distances in Å.

Figure 3

Comparison of cap-binding pockets. m7G-cap-binding pockets of CBP20, eIF4E and the viral nucleoside 2′-_O_-methyltransferase (VP39) are presented in comparison to the m3G-cap-binding pocket of snurportin1. Side chains of residues interacting with the caps are depicted in ball-and-stick mode. Atoms of the caps and the interacting side chains are coloured as described in Figure 1, with the exception of carbon atoms of the dinucleotide, which are shown in orange. In all presented cases, the residues stacking the bases and those forming hydrogen bonds with the cap bases are depicted. Hydrogen bonds are shown as dashed grey lines.

Figure 4

In vitro nuclear import assay of snurportin1. Nuclear import of fluorescently labelled U1snRNPs in the presence of importin-β, RanGDP, NTF2, an energy regenerating system, and either snurportin1 or one of the mutated proteins (W107A, W276A, and W107A/W276/A). In the left panel, cell nuclei stained with DAPI are shown. The middle panel shows the import of Cy3-labelled U1snRNPs. The quantification of the import rates is given in the right panel (a.u.: arbitrary units for intensity per pixel).

Figure 5

Structural similarity of snurportin1 with bound m3GpppG to the mRNA-guanylyltransferase with bound GTP. (A) Superimposed protein structures presented as ribbon diagrams and the bound nucleotides in ball-and-stick representation. Human snurportin1–m3GpppG complex (coloured red) and the mRNA-guanylyltransferase–GTP complex (coloured grey) of the Paramecium bursaria chlorella virus 1 (PDB accession code 1CKM) share an amino-acid sequence identity of 8.3% for the structurally homologous regions. (B) Close-up view of the nucleotide-binding pockets. In the mRNA-guanylyltransferase, the bound GTP protrudes much deeper into the cleft between the β-sheets. (C) Structure-based sequence alignment of human snurportin1 and human mRNA-guanylyltransferase reveals a sequence identity of 12.7%.

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Structural basis for m3G-cap-mediated nuclear import of spliceosomal UsnRNPs by snurportin1 - PubMed (original) (raw)