Journal Article

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

Ficner, Ralf, Dickmanns, Achim, Strasser, Anja, Lührmann, Reinhard

2005

Overview

In higher eukaryotes the biogenesis of spliceosomal UsnRNPs involves a nucleocytoplasmic shuttling cycle. After the m 7 G‐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 m 7 G‐cap is hypermethylated to the 2,2,7‐trimethylguanosine (m 3 G)‐cap. The import adaptor snurportin1 recognises the m 3 G‐cap and facilitates the nuclear import of the UsnRNPs by binding to importin‐β. Here we report the crystal structure of the m 3 G‐cap‐binding domain of snurportin1 with bound m 3 GpppG at 2.4 Å 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 m 7 G‐cap‐binding proteins Cap‐binding protein 20 (CBP20), eukaryotic initiation factor 4E (eIF4E) and viral protein 39 (VP39). The specificity of the m 3 G‐cap recognition by snurportin1 was evaluated by fluorescence spectroscopy, demonstrating the importance of a highly solvent exposed tryptophan for the discrimination of m 7 G‐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.

• Publisher: John Wiley & Sons, Ltd,Nature Publishing Group UK,Springer Nature B.V
• Subject: 5′-cap / Active Transport, Cell Nucleus / Amino Acid Sequence / Animals / Binding Sites / crystallography / Crystallography, X-Ray / EMBO36 / EMBO40 / Eukaryotic Initiation Factor-4E - chemistry / Fluorescence / HeLa Cells / Humans / Methylation / Models, Molecular / Molecular Sequence Data / Nuclear Cap-Binding Protein Complex - chemistry / Nuclear Localization Signals - chemistry / nuclear transport / Nucleotidyltransferases - chemistry / Protein Conformation / Proteins / Receptors, Cytoplasmic and Nuclear - chemistry / Receptors, Cytoplasmic and Nuclear - genetics / Ribonucleoproteins, Small Nuclear - chemistry / RNA Cap-Binding Proteins - chemistry / RNA Cap-Binding Proteins - genetics / RNA Caps - chemistry / snurportin1 / Solvents / Spectrometry, Fluorescence / Spectroscopy / Spliceosomes - chemistry / Tryptophan - chemistry / UsnRNP / Viral Proteins - chemistry