FIGURE 2.

Ysh1p is required for cleavage and polyadenylaion of pre-mRNA in vitro. (A, upper panel) In vitro cleavage and (lower panel) polyadenylation assays with protein extracts prepared from wild-type and ysh1 temperature-sensitive strains as indicated. Input shows a control where no protein was added. Positions of substrate RNAs, 5′ and 3′ end cleavage products and polyadenylation products bands are shown. HpaII-digested pBR322 fragments were 5′ end labeled and served as markers. Internally [³²P]-labeled substrate RNAs, CYC1 for the cleavage assay and CYC1-pre for specific polyadenylation, were used. Reactions were performed either at 30°C (lanes 1–6) or at 37°C (lanes 7–12). Extracts were preincubated at restrictive temperatures for 10 min prior to assaying. (B) As in A, except reactions were performed either at the permissive temperature 30°C (lanes 1–3) or at the non-permissive temperatures 17°C (lanes 4–6) for cleavage, and at 15°C for polyadenylation. (C,D) Reconstitution of specific cleavage and polyadenylation activities of the ysh1-32 extract in vitro. (C) Cleavage and (D) polyadenylation assays were performed essentially as in A, with protein extracts prepared from wild-type and the ysh1-32 strain as indicated. The ysh1-32 extract was combined with purified CPF to restore the specific 3′end processing activity. Twice more CPF was used for the reconstitution of polyadenylation than for the reconstitution of cleavage.