FIG. 6.

Knockdown of ubc9 by RNAi results in loss of cleavage and polyadenylation activity. (A) ³²P-labeled pG3SVL-A pre-RNA was incubated under standard cleavage conditions in NEs prepared from control siRNA-treated HeLa cells or ubc9 siRNA-treated cells. RNAs were resolved by denaturing PAGE. The unprocessed pre-RNA (SVL) and the 5′ and 3′ cleavage products are indicated. (B) ³²P-labeled pG3SVL-A precleaved pre-mRNA substrate (pre-SVL) was incubated as described for panel A under polyadenylation conditions, and RNAs were resolved by denaturing PAGE. The pre-mRNA substrate (precleaved SVL) and the polyadenylated products are indicated. (C) NEs made from control siRNA-treated cells (lane 1) or ubc9 siRNA-treated cells (lane 2) were analyzed by Western blotting with the antibodies indicated on the right. (D) HeLa cells were transfected with control siRNA (lane 1) or ubc9 siRNA (lane 2) and harvested, and extracts analyzed by Western blotting with antisymplekin (top panel), anti-ubc9 (middle panel), or antiactin (bottom panel) antibodies. The higher-molecular-weight forms of symplekin and the unmodified form are indicated by a bracket and an arrow, respectively. WB, Western blot.