title: Structural basis for m3G-cap-mediated nuclear import of spliceosomal UsnRNPs by snurportin1.
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.
authors.affiliation: Department of Molecular Structural Biology, Institute for Microbiology and Genetics, University Göttingen, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Strasser
authors.forename: Achim
authors.initials: A
authors.lastname: Dickmanns
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lührmann
authors.forename: Ralf
authors.initials: R
authors.lastname: Ficner
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D039561:Eukaryotic Initiation Factor-4E / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D039502:Nuclear Cap-Binding Protein Complex / Q000737:chemistry; D019913:Nuclear Localization Signals / Q000737:chemistry*; D009713:Nucleotidyltransferases / Q000737:chemistry; D011487:Protein Conformation; D039381:RNA Cap-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D012315:RNA Caps / Q000737:chemistry*; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D017381:Spliceosomes / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry; D000098556:mRNA Guanylyltransferases
chemical_list: D039561:Eukaryotic Initiation Factor-4E; D039502:Nuclear Cap-Binding Protein Complex; D019913:Nuclear Localization Signals; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps; D018160:Receptors, Cytoplasmic and Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C114090:SNUPN protein, human; C088682:VP39 protein, Vaccinia virus; D014764:Viral Proteins; D014364:Tryptophan; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases