A hallmark of metazoan RNA polymerase II transcripts is the presence of numerous 
small exons surrounded by large introns. Abundant evidence indicates that 
splicing to excise introns occurs co-transcriptionally, prior to release of the 
nascent transcript from RNAP II. Here, we established an efficient model system 
for co-transcriptional splicing in vitro. In this system, CMV-DNA constructs 
immobilized on beads generate RNAP II transcripts containing two exons and an 
intron. Consistent with previous work, our data indicate that elongating nascent 
transcripts are tethered to RNAP II on the immobilized DNA template. We show 
that nascent transcripts that reach full length, but are still attached to RNAP 
II, are efficiently spliced. When the nascent transcript is cleaved within the 
intron using RNase H, both the 5' and 3' cleavage fragments are detected in the 
bound fraction, where they undergo splicing. Together, our work establishes a 
system for co-transcriptional splicing in vitro, in which the spliceosome 
containing the 5' and 3' exons are tethered to RNAP II for splicing.