FIG. 4.

Snu17p is required for efficient splicing in vivo and in vitro. (A) RNA was extracted from wild-type (WT) and snu17Δ cells grown at 25°C (lanes 1 and 3) and after the switch to 37°C for 2 h (lane 4) and 4 h (lanes 2 and 5). Primer extension analysis was performed to measure the levels of unspliced pre-U3A and pre-U3B transcripts, indicated on the right. (B) Splicing reactions were performed at 25°C using the wild-type (lanes 1 to 8) and snu17Δ (lanes 9 to 16) extracts for the time indicated above each lane. To restore splicing, 1.2 pmol of recombinant GST-Snu17p was added to the reaction mixtures prior to addition of the actin precursor (lanes 5 to 8 and 13 to 16). Indicated on the left is the identity of the ³²P-labeled RNA species (from top to bottom): intron-lariat-exon 2 intermediate, excised lariat-intron, pre-mRNA, mature mRNA, and cleaved exon 1 intermediate.