Abstract
In metazoan organisms, pre-mRNA splicing is thought to occur during transcription, and it is postulated that these two processes are functionally coupled via still-unknown mechanisms. Current evidence supports co-transcriptional spliceosomal assembly, but there is little quantitative information on how much splicing is completed during RNA synthesis. Here we isolate nascent chromatin-associated RNA from free, nucleoplasmic RNA already released from the DNA template. Using a quantitative RT-PCR assay, we show that the majority of introns separating constitutive exons are already excised from the human c-Src and fibronectin pre-mRNAs that are still in the process of synthesis, and that these introns are removed in a general 5'-to-3' order. Introns flanking alternative exons in these transcripts are also removed during synthesis, but show differences in excision efficiency between cell lines with different regulatory conditions. Our data suggest that skipping of an exon can induce a lag in splicing compared to intron removal under conditions of exon inclusion. Nevertheless, excision of the long intron encompassing the skipped exon is still completed prior to transcript release into the nucleoplasm. Thus, we demonstrate that the decision to include or skip an alternative exon is made during transcription and not post-transcriptionally.