Figure 2.

Identification of U2AF⁶⁵, PTB, and SR proteins bound to the BPV-1 ESS. (A) ESS RNAs used for protein binding assays. The sequences of the ESS RNAs are shown, with capital letters indicating BPV-1 sequences, lowercase letters indicating the flanking sequences from the T7 promoter region, and solid lines indicating internal deletions. (B) The 5′ U-rich region of the ESS binds two proteins in HeLa NE. ³²P-rGTP-labeled RNAs A, B, and C, and ³²P-rUTP-labeled RNAs D and H were used for UV cross-linking of HeLa NE (20 μg) and digested with RNase T2 before electrophoresis. (C) IP by mAb 16H3 of a 65-kDa protein cross-linked to the 5′ U-rich region of the ESS. ESS RNAs E, F, G, and H were uniformly labeled with all four ³²P-rNTPs, UV cross-linked to the proteins in HeLa NE, digested with RNase A, and immunoprecipitated by using mAb 16H3 (33), anti-BPV-1 L1, or 20% fetal bovine serum (FBS) as indicated at the top. (D) Identification of the 65-kDa protein bound to the ESS as U2AF⁶⁵ (arrow) by using polyclonal anti-U2AF³⁵ antibody. ESS RNA G labeled with ³²P-rUTP (lanes 1–3) or all four ³²P-rNTPs (lanes 4–9) was UV cross-linked to proteins in either HeLa NE (lanes 1–4, 6, 8, and 9) or HeLa NE immunodepleted with anti-U2AF³⁵ (lane 5) or anti-U2AF⁶⁵ (lane 7). After digestion for 30 min with RNase A at 37°C, the RNA-protein mixtures were immunoprecipitated by using 20% FBS (lane 2), anti-U2AF³⁵ (lane 3), mAb 16H3 (lanes 4–7), anti-U2AF⁶⁵ (lane 8), or anti-BPV-1 L1 (lane 9) before loading for electrophoresis. Lane 1 is the UV cross-linked RNA-protein mixture without IP. (E) Identification of PTB bound to the 5′ U-rich region of the ESS by IP by using mAb 7G12. ESS RNA A labeled with ³²P-rGTP was used for UV cross-linking of HeLa NE followed by digestion with RNase T2. The cross-linked RNA-protein mixtures were immunoprecipitated by using anti-U2AF³⁵, anti-PTB mAb 7G12 (38), or 20% FBS. (F) Binding of recombinant PTB to the 5′ U-rich region of the ESS. HeLa NE (20 μg), poly (U)-depleted HeLa NE (NED) (20 μg), and/or recombinant PTB (1 μg) (30, 31) were used for UV cross-linking of ESS RNAs A, H, and C labeled with ³²P-rGTP. After digestion with RNase T2, the cross-linked RNA-protein mixtures were resolved by electrophoresis. (G–I) Identification of SR proteins bound to the BPV-1 ESS by IP. HeLa SR proteins (20 μg) were cross-linked by UV irradiation to RNAs E, F, G, or H labeled with ³²P-rGTP, digested with RNase A, and then resolved directly on a 12% SDS/PAGE gel (G, no IP) or subjected to IP by using mAb 1H4 (H) or mAb αASF2/SF2 (I) before electrophoresis. FBS (20%) was used as a mock antibody in both H and I.