G-TRIPLET LOCATED THROUGHOUT A CLASS OF SMALL VERTEBRATE INTRONS ENFORCE INTRON BORDERS AND REGULATE SPLICE-SITE SELECTION

Splicing of small introns in lower eucaryotes can be distinguished fro m vertebrate splicing by the inability of such introns to be expanded and by the inability of splice site mutations to cause exon skipping-p roperties suggesting that the intron rather than the exon is the unit of recognition. Vertebrates do contain small introns. To see if they p ossess properties similar to small introns in lower eucaryotes, we stu died the small second intron from the human cy-globin gene. Mutation o f the 5' splice site of this intron resulted in in vivo intron inclusi on, not exon skipping, suggesting the presence of intron bridging inte ractions. The intron had an unusual base composition reflective of a s equence bias present in a collection of small human introns in which m ultiple G triplets stud the interior of the introns. Each G triplet re presented a minimal sequence element additively contributing to maxima l splicing efficiency and spliceosome assembly. More importantly, G tr iplets proximal to a duplicated splice site caused preferential utiliz ation of the 5' splice site upstream of the triplets or the 3' splice site downstream of the triplets; i.e., sequences containing G triplets were preferentially used as introns when a choice was possible. Thus, G triplets internal to a small intron have the ability to affect spli ce site decisions at both ends of the intron. Each G triplet additivel y contributed to splice site selectivity. We suggest that G triplets a re a common component of human 5' splice sites and aid in the definiti on of exon-intron borders as well as overall splicing efficiency. In a ddition, our data suggest that such intronic elements may be character istic of small introns and represent an intronic equivalent to the exo n enhancers that facilitate recognition of both ends of an exon during exon definition.