Structure of the U6 RNA intramolecular stem-loop harboring an S sub(P)-phosphorothioate modification

Phosphorothioate-substitution experiments are often used to elucidate functionally important metal ion-binding sites on RNA. All previous experiments with S sub(P)-phosphorothioate-substituted RNAs have been done in the absence of structural information for this particular diastereomer. Yeast U6 RNA contains a metal ion-binding site that is essential for spliceosome function and includes the pro-S sub(P) oxygen 5' of U sub(80). S sub(P)-phosphorothioate substitution at this location creates spliceosomes dependent on thiophilic ions for the first step of splicing. We have determined the solution structure of the U sub(80) S sub(P)-phosphorothioate-substituted U6 intramolecular stem-loop (ISL), and also report the refined NMR structure of the unmodified U6 ISL. Both structures were determined with inclusion of super(1)H- super(13)C residual dipolar couplings. The precision of the structures with and without phosphorothioate (RMSD = 1.05 and 0.79 Ae, respectively) allows comparison of the local and long-range structural effect of the modification. We find that the U6-ISL structure is unperturbed by the phosphorothioate. Additionally, the thermodynamic stability of the U6 ISL is dependent on the protonation state of the A sub(79)-C sub(67) wobble pair and is not affected by the adjacent phosphorothioate. These results indicate that a single S sub(P)-phosphorothioate substitution can be structurally benign, and further validate the metal ion rescue experiments used to identify the essential metal-binding site(s) in the spliceosome.