RNA polymerase II (RNAP II) is responsible for transcription of mRNA precur
sors in eukaryotic cells. Recent studies, however, have suggested that RNAP
II also participates in subsequent RNA processing reactions through intera
ctions between the carboxy-terminal domain (CTD) of the RNAP II largest sub
unit and processing factors. Using reconstituted in vitro splicing assays,
we show that RNAP II functions directly in pre-mRNA splicing by influencing
very early steps in assembly of the spliceosome. We demonstrate that the p
hosphorylation status of the CTD dramatically affects activity: Hyperphosph
orylated RNAP IIO strongly activates splicing, whereas hypophosphorylated R
NAP IIA can inhibit the reaction.