Many proteins involved in pre-mRNA processing contain one or more copies of a 70-90-amino-acid alphabeta module called the ribonucleoprotein domain. RNA maturation depends on the specific recognition by ribonucleoproteins of RNA elements within pre-mRNAs and small nuclear RNAs. The human U1A protein binds an RNA hairpin during splicing, and regulates its own expression by binding an internal loop in the 3'-untranslated region of its pre-mRNA, preventing polyadenylation. Here we report the nuclear magnetic resonance structure of the complex between the regulatory element of the U1A 3'-untranslated region (UTR) and the U1A protein RNA-binding domain. Specific intermolecular recognition requires the interaction of the variable loops of the ribonucleoprotein domain with the well-structured helical regions of the RNA. Formation of the complex then orders the flexible RNA single-stranded loop against the protein beta-sheet surface, and reorganizes the carboxy-terminal region of the protein to maximize surface complementarity and functional group recognition.
The N-terminal RNP domain of U1A binds two different RNA substrates with high affinity and specificity: stem-loop II of the U1 snRNA and a complex secondary structure in the 3'-untranslated region (3'-UTR) of the U1A pre-mRNA. Both RNAs contain a single-stranded sequence which is the main site of interaction with the protein, but in completely different structural contexts. Here we describe the solution structure of the free 3'-UTR RNA molecule and the NMR characterization of its complex with the U1A protein N-terminal domain. The structure of the free RNA indicates that the stems are nearly canonical A-form helices and that the single-stranded region contains local stacking interactions in the context of a generally flexible structure. Upon protein binding, the internal loop region folds into an ordered structure containing significant changes in the local stacking interactions. These results demonstrate the role of RNA structure and folding in specific RNA-protein recognition.
Backbone-driven assignment methods that utilize covalent connectivities have greatly facilitated spectral assignments of proteins. In nucleic acids, 1H-13C-31P correlations could play a similar role, and several related experiments (HCP) have recently been presented for backbone-driven sequential assignments in RNA. The three-dimensional extension of 1H-31P Het-Cor (P,H-COSY-H,C-HMQC) and Het-TOCSY (P,H-TOCSY-H,C-HMQC) experiments presented here complements HCP experiments as tools for spectral assignments and extraction of dihedral angle constraints. By relying on 1H-31P rather than 13C-31P couplings to generate cross peaks, the strongest connectivities are observed in different spectral regions, increasing the likelihood of resolving spectral overlap. In addition, semiquantitative estimates of 1H-31P and 13C-31P couplings provide dihedral angle constraints for RNA structure determination.
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