FIG. 4.

Analysis of in vitro coupled cleavage and polyadenylation reactions in mex67-5 extract. (A) Extract from mex67-5 cells processes RNA efficiently but gives longer poly(A) tails than that of WT extract. Cells were grown in liquid YPD at 25°C and then shifted to 37°C for 1 h before extracts were prepared. Processing reactions were performed by incubation with a radioactively labeled precursor RNA containing the GAL7 poly(A) site (Pre) at 30°C for the indicated times. (B) Tails are not shortened in mex67-5 extract if processing is performed at 37°C instead of 30°C. The slight decrease in processing efficiency in the mex67-5 extract at 37°C in this experiment is not reproducible. (C) The PAN nuclease trims tails synthesized in vitro. Extracts were prepared from WT and mex67-5 cells expressing tandem affinity purification-tagged Pan2p, and PAN was removed by incubation with IgG beads. (Left) Western blotting analysis of Pan2 in mock-depleted (+) or PAN-depleted (−) extract. (Right) Processing in extracts with or without PAN. Reactions were performed for 120 min at 30°C. (D) The mex67-5 mutation does not cause an increased association of Rna15p with RNAs that are polyadenylated in vitro. Processing reaction mixtures were incubated at 30°C for 30 min, and RNAs were immunoprecipitated using Rna15p antibody (+) or preimmune serum (−).