In order to examine whether splicing can occur cotranscriptionally in mammalian nuclei, we mapped exon-intron boundaries on nascent RNA chains transcribed by RNA polymerase II. A procedure that allows fractionation of nuclei into a chromatin pellet containing DNA, histones, and ternary transcription complexes and a supernatant containing the bulk of the nonhistone proteins and RNAs that are released from their DNA templates was developed. The transcripts of the genes encoding DBP, a transcriptional activator protein, and HMG coenzyme A reductase recovered from the chromatin pellet and the supernatant were analyzed by Si nuclease mapping. The large majority of the RNA molecules from the pellet appeared to be nascent transcripts, since, in contrast to the transcripts present in the supernatant, they were not cleaved at the polyadenylation site but rather contained heterogeneous 3' termini encompassing this site. Splicing intermediates could be detected among nascent and released transcripts, suggesting that splicing occurs both cotranscriptionally and posttranscriptionally. Our results also indicate that polyadenylation is not required for the splicing of the last DBP intron. In addition to allowing detailed structural analysis of nascent RNA chains, the physical isolation of nascent transcripts also yields reliable measurements of relative transcription rates.In higher eukaryotes, the large majority of mRNAs are synthesized as large precursor mRNAs (pre-mRNAs) containing multiple intervening sequences. These sequences are subsequently removed by a process called splicing before the mRNA is transported from the nucleus to the cytoplasm (for reviews see references 11 and 19). As revealed by microscopy studies, actively transcribed genes, polyadenylated RNAs, and components of the splicing machinery appear to colocalize in multiple small subcompartments of the cell nucleus (13,32 released from their templates indicate that splicing occurs both cotranscriptionally and posttranscriptionally.
MATERUILS AND METHODSRun-on transcription in isolated nuclei. Liver cell nuclei were isolated from adult male rats (Lewis strain) as described by Lichtsteiner et al. (18) and resuspended in a solution containing 20 mM Tris (pH 7.9), 75 mM NaCl, 0.5 mM EDTA, 0.85 mM dithiothreitol (DTT), 0.125 mM phenylmethylsulfonyl fluoride, 0.1 mg of yeast tRNA per ml, and 50% glycerol, at a concentration of 105 nuclei per ,l. The elongation of nascent RNA chains was performed as described by Schibler et al. (27), with the following modifications. Nascent RNAs were elongated in the absence of (NH4)2SO4 and heparin sulfate. The elongation buffer contained 0.2 ,uM (instead of 5 ,uM) [a-32P]UTP (400 Ci/mmol) and 1 mM unlabeled UTP. The elongation was performed for 0, 2, and 5 min at 26°C. The reaction was stopped by the addition of a solution containing 10 volumes of ice-cold 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH 7.6), 0.2 mM EDTA, 7.5 mM MgCl2, 1 mM DTT, 0.1 mg of yeast tRNA per ml, 0.3 M NaCl, 1% Nonidet P-40, and ...