Recognition of Cap Structure in Splicing in Vitro of MRNA Precursors

Substrate RNAs are only efficiently spliced in HeLa whole-cell extract when they possess capped 5' termini. This cap requirement is observed with substrate RNAs prepared by transcription with either mammalian RNA polymerase II or bacterial RNA polymerase. Addition of less than 10 microM of cap analogs such as m7G(5')ppp(5')N or m7GTP strongly inhibits splicing of capped RNAs. This observation, as well as experiments following the fate of substrate RNA, indicates that the dependence of splicing on a cap structure is not due to an effect on RNA stability. More interestingly, cap analogs inhibit splicing when added at the start of the reaction but not at later times of incubation. This suggests that the cap recognition might be an important step in the formation of a specific ribonucleoprotein complex required for splicing.