β-globin transcripts carrying a single intron with three adjacent nucleotides of 5′ exon are efficiently splicedin vitroirrespective of intron position or surrounding exon sequences

To examine the role of exon sequences and intron position in the splicing of an mRNA precursor, we prepared series of sense or anti-sense transcripts of human beta-globin cDNA in which a cassette containing the beta-globin first intron was inserted into one of seven unusual positions. The intron cassette consisted of the intron alone (ml), the intron with three adjacent base pairs of the 5' exon (MI), or the intron with both 5' and 3' exon sequences. All these transcripts were examined in an in vitro splicing system with a HeLa cell nuclear extract. The sense transcripts carrying MI cassette were spliced efficiently and independently of the intron position, except when the 3' exon was too short. The anti-sense transcripts carrying MI cassette produced significantly less spliced products than did those of the sense transcripts. This was mostly because of the instability of the anti-sense transcripts, and the actual splicing efficiency was similar to that seen in the sense transcripts. Sense or anti-sense transcripts carrying ml cassette were spliced to various extents depending on the surrounding sequences. The results indicate that only three nucleotides of the 5' exon are required as specific exon sequences in the splicing of an mRNA precursor carrying a single intron, and that the intron position does not significantly affect the splicing efficiency in vitro.