T. thermophilus TruD pseudouridylates CDC8 mRNA fragments in vitro. (A) Secondary structure of the CDC8 mRNA fragment with the recognition sequence of TruD in green. (B) In vitro pseudouridine formation assays using recombinant TruD protein and the CDC8 fragment. (C) Single-point mutations in the CDC8 fragment with neutral (blue) or disruptive (red) effect on pseudouridylation. (D–N) In vitro pseudouridylation assays with (plus TruD) or without (minus TruD) recombinant TruD protein using indicated CDC8 mutants (D) U15C, (E) U13A, (F) U13C, (G) G14A, (H) G14C, (I) G14U, (J) A16G, (K) G17C, (L) G17A, (M) G17C, and (N) U12C. These experiments were performed independently two times (n = 3) and gave the same results; one representative chromatogram was shown. (O) T. thermophilus TruD recognizes the UNUAR sequence in substrate RNAs with the target uridine underlined and highlighted in blue in (N), where N is any nucleotide (A, U, G, C), and R is purine residues (A, G).
