Fig. 6.

Site-specific cross-linking experiments. (A) Precursor RNAs containing a single ³²P (marked by *) and an s⁴U residue (indicated by a lightning bolt) four nucleotides downstream of the ³²P label. (B) Splicing was carried out with the indicated precursors and aliquots of each mixture were analyzed by denaturing (7M urea) PAGE and autoradiography. (C) The samples were separated by SDS-PAGE after UV irradiation and RNase T1 digestion and ³²P-labeled polypeptides were visualized by autoradiography. The positions (in kDa) of pre-stained marker proteins are indicated at the right. (D) Singly-labeled pre-mRNAs with (+) or without (−) s⁴U at position +17, were spliced in wild-type extract supplemented with wild-type Prp22 or the dominant-negative Prp22-T765A protein. The RNA species were analyzed by denaturing PAGE (left panel) and the ³²P-labeled polypeptides were visualized by SDS-PAGE and autoradiography after cross-linking and RNase T1 digestion (right panel). (E) Pre-mRNA containing a single ³²P, and s⁴U at position +17 was spliced in wild-type extract supplemented with Prp22, Prp22-T765A or Prp22-K512A-TAP proteins. After cross-linking and RNase T1 digestion, the samples were analyzed by SDS-PAGE and autoradiography. (F) s⁴U-containing +17 pre-mRNA was spliced in Δprp22 extract supplemented with Prp22-T765A or Prp22-N 260-T765A polypeptides. The reaction mixtures were exposed to UV, treated with RNase T1 and analyzed by SDS-PAGE. An autoradiogram of the dried gel is shown.