FIG. 1.

The PAN RNase is present in RNA 3′-end processing extracts. (A) Wild-type (WT) and Pan3p-deficient (panΔ) yeast S-100 extracts were ammonium sulfate precipitated as described in Materials and Methods. The total starting extracts (T), soluble fractions (S), and precipitatant fractions (P) were resolved by SDS-PAGE, and the indicated proteins were detected by Western blot analysis. Wild-type and Pan3p-deficient cell extracts were prepared from strains YAS306 and YAS1943, respectively. Equivalent percentages of the fractions were loaded onto the lanes. (B) Detection of PAN activity in 3′-end processing extracts prepared from pan and pab1 mutant yeast strains. One microgram of total protein was incubated with radiolabeled poly(A) and either recombinant Pab1p (black bars), pab1-55p (grey stippled bars), or no additional Pab1p (white bars) as described in Materials and Methods. Nuclease activity was measured by quantifying the release of trichloroacetic acid (TCA)-soluble [³²P]AMP (y axis). The purified PAN enzyme serves as a positive control for the Pab1p-stimulated RNase activity. The yeast strains used to prepare the 3′-end processing extracts were (from left to right) YAS306, YAS2283, YAS1943, YAS1942, YAS1254, and YAS1255. Abbreviations: recomb., recombinant; WT, wild type.