We have studied the involvement of the 5' cap structure in the splicing of precursor mRNAs in a HeLa nuclear extract. We show that precursor mRNAs are spliced efficiently only when they possess a cap structure and that preincubation of a HeLa nuclear extract rendered the splicing reaction highly sensitive to inhibition by cap analogues. This sensitization was dependent on exogenous Mg2' but not exogenous ATP or GTP.These results demonstrate that splicing in a nuclear extract is highly dependent on the cap structure as was demonstrated for the splicing process in a HeLa whole-cell extract [Konarska, M. M., Padget, R. A. & Sharp, P. A. (1984) Cell 38,[731][732][733][734][735][736], and thus support the contention that cap recognition is an important feature of eukaryotic mRNA biogenesis.All eukaryotic cellular mRNAs analyzed to date are blocked at their 5' terminus by the cap structure, m7G(5')ppp(5')N (1). There is considerable evidence that the cap structure enhances translational efficiency by facilitating ribosome binding to mRNA (1). This interaction is mediated by a distinct group of proteins defined as cap-binding proteins (2,3).With the advent of efficient in vitro splicing systems (4-10), two recent studies have suggested that the cap structure may play a significant role in the splicing process as well (6, 7). Using a HeLa nuclear extract, Krainer et al. (7) were able to show efficient splicing of in vitro synthesized, truncated human P-globin transcripts. They showed that although uncapped transcripts could still be spliced, the efficiency was one-half to one-third that for their enzymatically capped counterparts. In addition, splicing of uncapped precursor mRNAs (pre-mRNAs) The transcripts were stored at -70'C in water and used directly in the splicing reaction.In Vitro Splicing Reaction. HeLa nuclear extracts were prepared as described (13). All experiments described throughout were performed using the same extract prepara-
RESULTSTo characterize the splicing activity of our HeLa nuclear extracts, we used the plasmid pSP64-il/3A6. This plasmid and its use in the nuclear splicing system were described by Krainer et al. (7) and Ruskin et al. (8). For
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