Structure of the U6 RNA intramolecular stem–loop harboring an SP-phosphorothioate modification

Open Access
Publisher Website
Google Scholar
Abstract
Phosphorothioate-substitution experiments are often used to elucidate functionally important metal ion-binding sites on RNA. All previous experiments with SP-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-SP oxygen 5′ of U80. SP-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 U80SP-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 1H–13C residual dipolar couplings. The precision of the structures with and without phosphorothioate (RMSD = 1.05 and 0.79 Å, 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 A79–C67 wobble pair and is not affected by the adjacent phosphorothioate. These results indicate that a single SP-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.