Figure 3.

Reciprocal effects of SmB/B' and TUT7 on the cellular level of U2-tfs/U2-tfs-polyU. (A) SmB/B' stabilizes U2-tfs/U2-tfs-polyU. RNA extracted from cells transfected with a small interfering RNA (siRNA, si) or scrambled-sequence RNA (scRNA, sc, control) for the knockdown of SmB/B', Dis3L2, or both was analyzed with northern blotting using the probes shown to the left of the blot. An immunoblot (IB) is shown for SmB/B' or Dis3L2. Values shown under the protein blot indicates the staining intensity of each protein band relative to that the control (sc SmB/B', sc Dis3L2). (B) Deficiency of TUT7 increases the level of U2-tfs/U2-tfs-polyU. RNA extracted from the cells at 48 h and 72 h post-transfection with siRNA (or scRNA control) for knockdown of SmB/B', Dis3L2 or TUT7, or a combination of those siRNAs, was analyzed by northern blotting using the probes shown to the left side in right panel. Stained bands corresponding to U1-tfs, U2-tfs, and U2-tfs-polyU are shown to the right. Proteins were analyzed by immunoblotting with antibodies against the proteins indicated to the left side in left panel. (C) Northern blotting with probe U1-#1, U2-#1 or Met-tRNA (loading control) detected RNAs extracted from scRNA- or TUT7 siRNA–transfected cells treated with (+) or without (–) doxycycline inducing Dis3L2-D391N-FLAG expression. Protein was detected by immunoblotting with antibodies against the proteins indicated to the left.