PHYSICAL ISOLATION OF NASCENT RNA CHAINS TRANSCRIBED BY RNA-POLYMERASE-II - EVIDENCE FOR COTRANSCRIPTIONAL SPLICING

In order to examine whether splicing can occur cotranscriptionally in mammalian nuclei, we mapped exon-intron boundaries on nascent RNA chai ns transcribed by RNA polymerase II. A procedure that allows fractiona tion of nuclei into a chromatin pellet containing DNA, histones, and t ernary transcription complexes and a supernatant containing the bulk o f the nonhistone proteins and RNAs that are released from their DNA te mplates was developed. The transcripts of the genes encoding DBP, a tr anscriptional activator protein, and HMG coenzyme A reductase recovere d from the chromatin pellet and the supernatant were analyzed by S1 nu clease mapping. The large majority of the RNA molecules from the pelle t appeared to be nascent transcripts, since, in contrast to the transc ripts present in the supernatant, they were not cleaved at the polyade nylation site but rather contained heterogeneous 3' termini encompassi ng this site. Splicing intermediates could be detected among nascent a nd released transcripts, suggesting that splicing occurs both cotransc riptionally and posttranscriptionally. Our results also indicate that polyadenylation is not required for the splicing of the last DBP intro n. In addition to allowing detailed structural analysis of nascent RNA chains, the physical isolation of nascent transcripts also yields rel iable measurements of relative transcription rates.