AN INTRON SPLICING ENHANCER CONTAINING A G-RICH REPEAT FACILITATES INCLUSION OF A VERTEBRATE MICRO-EXON

The average length of a vertebrate exon is approximately 130 nt. Decre asing the size of an internal exon to less than 51 nt induces exon ski pping,implying a minimal size for exons. A few constitutively included internal exons, however, are extremely small. To investigate if such micro-exons require special mechanisms for their inclusion, we studied the sequences necessary for inclusion of a 6-nt exon from chicken car diac troponin T (cTNT). In vivo, the cTNT micro-exon was not included in mRNA unless accompanied by a 134-nt sequence located next to the mi cro-exon in the downstream intron. Increasing the length of the micro- exon alleviated the requirement for the intron element, indicating tha t the lack of inclusion of the micro-exon in the absence of a facilita ting sequence was due to its small size, rather than suboptimal splice sites. The intron element contained six copies of a G-rich 7-nt seque nce. Multimers of the repeat supported exon inclusion, indicating that the repeat sequence is an important part of the intron element. The e ntire intron element activated inclusion of a heterologous 7-nt exon, suggesting that the intron element is a general enhancer for the splic ing of micro-exons. In vitro, the intron element and the repeated sequ ence facilitated splicing of a heterologous exon. Because of the abili ty of the cTNT intron element to facilitate the splicing of heterologo us exons, we have termed the element an intron splicing enhancer (ISE) . Interestingly, the ISE demonstrated position independence in that it facilitated inclusion of the heterologous micro-exon when placed eith er upstream or downstream of the micro-exon. In vitro, the ISE or copi es of the ISE G-rich repeat stimulated splicing of an adjacent intron. The ISE th us becomes one of only a few characterized ISEs containing a G-rich repeat and the first to work both upstream and downstream of a target exon.