Figure 3. Efficient Nab2p incorporation into pA tail RNP only occurs in the absence of Rrp6p.

(A) CYC1s in vitro polyadenylation reactions carried out for 30 min in wt or rrp6Δ extracts were terminated by the addition of excess dATP and subjected to IP by magnetic beads containing no (mock), α-Pab1p or α-Nab2p antibody. RNA was collected from input, unbound and bead-bound fractions and resolved by denaturing PAGE. Quantification of relative IP efficiencies of mature and hyperadenylated RNA species (see Experimental Procedures) are shown below the images labeled as in Figure 2A. The first lane (not numbered) was loaded with an equivalent amount of CYC1s precursor.

(B) Same as in (A) but using trf4Δ and rrp6Δ/trf4Δ extracts for polyadenylation reactions.

(C) Pab1p and Nab2p association with Cbp20p-bound RNPs in vivo. Cbp20p-TAP was purified using magnetic beads coated with rabbit IgG from wt or rrp6Δ cells and eluates were examined by western blotting analysis for their contents of Cbp20p-TAP, Cbp80p, Pab1p and Nab2p. ‘No TAP’ denotes a negative control strain carrying un-tagged Cbp20p. 0.5% of the total input (lanes 1–3) was loaded next to IP reactions carried out without (lanes 4–6) or with (lanes 7–9) addition of an RNase cocktail. Longer exposures of Cbp20p-TAP and Cbp80p blots are shown to reveal the presence of these proteins in input lanes.

See also Figure S2.