FIGURE 6.

Effect of mutations in the RPL30 intron and Cbp80 on cotranscriptional recruitment of U1 and U2 on the RPL30-LacZ gene. Horizontal axes show the distance in nucleotides from the start codon. Vertical axes indicate the signal relative to that of the promoter (first primer pair, or PP1). The black bar indicates intron position. The ChIP profiles correspond to wt (black lines) or cbp80Δ cells (gray lines). (A) Panels indicate different intronic 5′ ends, with a schematic representation of the possible base-pairing with U1 snRNA. GUCAGUAU panels are based on data from Figure 5. (B–D) ChIP profiles of U1 snRNP (Snu71-HTB). (E–G) ChIP profiles of U2 snRNP (Lea1-HTB). (H,I) In vitro splicing of the RPL30 intron, either wt or with reduced affinity for U1 snRNA, in wt or cbp80Δ extracts. Splicing reactions were set up and analyzed as in Figure 4, using wt extracts (H) or extracts from cbp80Δ cells (I), supplemented with MBP:L30 or MBP:Cbp80, as indicated. In the UC transcript, intron positions 6 and 7 (AU) have been mutated to UC, which cannot base-pair to U1. Bands corresponding to substrate and spliced RNA are shown. Amounts of RNA, extracts, and recombinant protein were equivalent in all reactions (see Materials and Methods).