Figure 1.

Cleavage activity of an intronic ribozyme occurs cotranscriptionally and is averted by the splicing pathway. (A) Four splicing reporter plasmids containing a single 5′ exon MS2 stem–loop were derived from the galactose-driven RP51a–lacZ fusion gene HZ18 (Teem and Rosbash 1983) with and without a 5′SS (GTATGT, Δ5′SS) and with either an active ribozyme (RZ [blue]) or a point mutation killing the RZ activity (mutRZ [red]). HA-MS2 ChIPs were performed as described (see Materials and Methods) on the wild-type reporter genes (GTATGT) with (RZ [blue]) and without (mutRZ [red]) an active ribozyme. The Y-axis represents fold enrichment relative to the first primer pair. The gene model shows the location of the MS2 site (purple), the intron (black line), the RZ sequences, and the lacZ coding sequence (rectangle), and is drawn to scale with the X-axis, which represents the distance from the start codon. The dashed line within the graph shows the level of background MS2 signal as measured by the amount of DNA bound from the PMA1 gene relative to the first primer pair. (B) MS2 ChIPs for the reporters lacking a 5′SS (Δ5′SS) with the active RZ (RZ [blue]) or inactive RZ (mutRZ [red]) show that RZ cutting occurs cotranscriptionally when splicing is abrogated. (C) The patterns of the RZ and mutRZ curves are very similar, suggesting that little cleavage activity is occurring before splicing. The schemes to the right of each graph display a pictorial interpretation of the data.