Identification of an RNA binding site for human thymidylate synthase

Previous studies from this laboratory have shown that human TS mRNA translation is regulated by its protein product in a negative autoregulatory manner. In this paper, we identify an RNA binding site for TS protein located within the first 188 nt of TS RNA. A 36-nt RNA sequence contained within this 188-nt fragment, corresponding to nt 75-110 and including the translational initiation site, binds TS protein with an affinity similar to that of both the full-length and the 188-nt TS RNA sequences. Variant RNAs with either a deletion or a mutation at the translational initiation region are unable to compete for TS protein binding. UV crosslinking studies reveal that an RNA fragment of ≃36 nt is protected from RNase T1 digestion by TS protein binding. A second TS protein-binding site is localized within the protein- coding region corresponding to nt 434-634. These findings demonstrate a specific interaction between human TS protein and its TS RNA and identify an RNA binding site that includes the translational initiation site.