IN-VITRO SPLICING OF PRE-MESSENGER-RNA CONTAINING BROMOURIDINE

The artificial UTP-analogue 5-bromouridine 5'-triphosphate (BrUTP) has been used to label pre-mRNA in vitro and in vivo [1, 2]. We have inve stigated the effect of bromouridine (BrU) in pre-mRNA on the efficienc y of splicing. An adenovirus major late II construct was used to prepa re four different transcripts, each containing a different amount of B rU. These four transcripts were tested in an in vitro splicing assay. We found that splicing is strongly inhibited if all uridines (U) in th e transcript were substituted for BrU. Splicing was restored to some e xtent if 50% of the Us were replaced by BrU. The splicing efficiency r eturned to an almost normal level if only 1 out of every 10 Us was sub stituted for BrU. This demonstrates that only a pre-mRNA containing a small amount of BrU can be spliced normally; in vitro. Furthermore, th ese results strongly suggest that some Us in the adenoviral transcript , probably those at the splice sites, cannot be replaced by BrU and ar e therefore critical in the splicing reaction.