FIG. 3.

Deadenylation by the PAN RNase is required for proper poly(A) tail length control in vitro. (A) Cleavage and polyadenylation reactions were performed and visualized as described in the legend to Fig. 2. RNA 3′-end processing extracts described in the legend to Fig. 1B were incubated with (+) or without (−) purified PAN. Lane 1, DNA markers (M); lane 2, input CYC1 pre-RNA (precursor [pre]); lane 3, product of cleavage reaction only. The migration positions of the 5′-upstream cleavage product and the polyadenylated products are indicated to the right, and the sizes (in nucleotides) of the DNA markers are indicated to the left. WT, wild type. (B) Time course of CYC1 3′-end processing was carried out in either wild-type extracts of strain YAS306 (lanes 3 to 7), pan3Δ mutant extracts of YAS1943 (lanes 8 to 12), or pan3Δ mutant extracts, containing exogenously added purified PAN (lanes 13 to 21). The purified enzyme was either added at the beginning of the reaction (lanes 13 to 17) or 60 min post-CYC1 cleavage and polyadenylation (lanes 18 to 21). Cleavage and polyadenylation reactions were carried out and visualized as described in the legend to Fig. 2. Lane 1, DNA markers (M); lane 2, input CYC1 pre-RNA (precursor [pre]). The migration positions of the 5′-upstream cleavage product and the polyadenylated products are indicated to the right, and the sizes (in nucleotides) of the DNA markers are indicated to the left.