INVERSE SPLICING OF A DISCONTINUOUS PRE-MESSENGER-RNA INTRON GENERATES A CIRCULAR EXON IN A HELA-CELL NUCLEAR EXTRACT

We have recently reported the first example of inverse splicing of a e ukaryotic pre-mRNA intron using a whole cell extract from the yeast Sa ccharomyces cerevisiae. The concomitant circularization of the exon in the course of this splicing reaction gave rise to the hypothesis that the circular RNA species, which had been recently discovered in vivo in mammalian cells, were generated by inverse splicing, Here we report the formation of a circular exon in HeLa cell nuclear extracts by an inverse splicing reaction of the second intron of the human beta-globi n gene from a pre-mRNA transcript in which the two intron halves flank ed an artificially fused, single exon. Our data demonstrate that the m ammalian pre-mRNA splicing system has indeed an intrinsic capability o f aligning splice sites in reverse order and that this alignment can b e followed by a complete splicing reaction, whereby the discontinuous intron sequences are removed. Thus we propose that circular exons in v ivo arise as a result of an inverse splicing reaction following the pa iring of a 5' splice site with an upstream 3' splice site and that the frequency of this event is influenced by the presence and strength of other, competing splice sites.