NMR studies of U1 snRNA recognition by the N-terminal RNP domain of the human U1A protein.
The RNP domain is a very common motif found in hundreds of proteins, including many protein components of the RNA processing machinery. The 70-90 amino acid domain contains two highly conserved stretches of 6-8 amino acids (RNP-1 and RNP-2) in the central strands of a four-stranded antiparallel beta-sheet, packed against two alpha-helices by a conserved hydrophobic core. Using multidimensional heteronuclear NMR, we have mapped intermolecular contacts between the human U1A protein 102 amino acid N-terminal RNP domain and a 31-mer oligonucleotide derived from stem-loop II of U1 snRNA. Chemical shift changes induced on the protein by the RNA define the surface of the beta-sheet as the recognition interface. The reverse face of the protein, with the two alpha-helices, remains exposed to the solvent in the presence of the RNA, and is potentially available for protein-protein contacts in spliceosome assembly or splice site selection. Protein-RNA contacts occur at the single-stranded apical loop of the hairpin, but also in the major groove of the helical stem at neighbouring U.G and U.U non-Watson-Crick base pairs. Examination of a proposed model for the complex in the light of the present results reveals several features of RNA recognition by RNP proteins. The quality of the spectra for this complex of 22 kDa demonstrates the feasibility of NMR investigation of RNA-protein complexes.
Howe
P W
PW
MRC Laboratory of Molecular Biology, Cambridge, UK.
Nagai
K
K
Neuhaus
D
D
Varani
G
G
D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012342:RNA, Small Nuclear / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism*; D013329:Structure-Activity Relationship
D009841:Oligonucleotides; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein