Figure 7.

Differential effect of interspersed non-A nucleotides on deadenylation by the CCR4-CAF1 complex. (A) Discrimination against non-A nucleotides in the active sites of CCR4 (+/−) and CAF1 (−/+). Position A16 of the ^5FN₇A₂₀ RNA substrate was substituted with a pyrimidine (uridine or cytidine; substrate denoted as ^5FN₇A₈UA₁₁ or ^5FN₇A₈CA₁₁) or another purine (guanine; substrate denoted as ^5FN₇A₈GA₁₁). (B) Discrimination against inosine. Position A16 of the ^5FN₇A₂₀ RNA substrate was substituted with an inosine, the direct deamination product of adenine (substrate denoted as ^5FN₇A₈IA₁₁). Panels (A) and (B) show comparative gels with samples selected and reorganized from Supplementary Figure S8. Note the diversity and the precise position of each of the primary product bands in the 2-fold vertical zoom onto the 16 nt marker region at the bottom of each panel. Importantly, and depending on the individual structural context, the non-A nucleotides are frequently rate-limiting already before they reach the 3′-terminal position in the RNA substrate, occasionally even from the pre-penultimate position. (C) Effects of non-A nucleotides on deadenylation. The scheme highlights phosphodiester linkages where the rate of hydrolysis by CCR4 or CAF1 is reduced, and where the number of blunt arrowheads indicates the extent of the reduction.