Figure 4.

Box C/D and C'/D' are functionally independent. (A, B) Secondary structure of archaeal sR1c (A) and eukaryotic Ct snR61 (B) RNAs. The analyzed mutations (mut) and deletions (Δ) are illustrated. The locations of m⁶A modification in box D and D' of snR61 are marked. (C, D) Modification activity of C/D RNPs assembled with sR1c (C) and snR61 (D) mutant RNAs. Ct RNPs were assembled with either C/D RNA (2 μM), Snu13 (6 μM), Nop56/Fib (2 μM) and Nop58/Fib (2 μM). Ss RNPs were assembled with either C/D RNA (1 μM), co-purified Nop5/Fib complex (2 μM) and L7Ae (3 μM). The D and D' substrates and mixed pre-methylated substrates (sub-m) were reacted for 20 min by Ct RNPs at 50°C and by Ss RNPs at 70°C. The relative level of modification shown under each band was normalized to that guided by wild-type sR1c or snR61.