FIGURE 1.

AG dinucleotide exclusion zones and distant branch points. (A) The branch point sequence (BPS), polypyrimidine tract (PPT), and the invariant AG at the 3′ ss are normally located within 40 nt of each other. The region between the 3′ ss AG and the next upstream AG dinucleotide is defined as the AG exclusion zone (AGEZ). In introns with distant BPs, there is no apparent constraint on the distance between the PPT and 3′ ss as long as it is free of AGs. (B) Four exons of mammalian serotonin receptor (HTR4) genes have extended AGEZ. The position of the first AG upstream of the 3′ ss is shown for each exon in five different vertebrates. For dog and chicken, the genomic sequence coverage is not sufficient to determine their AGEZ of exon g (n/a) and in rat only the alternative 3′ ss variant e is present (*). (C) HTR4 isoforms: Exons are depicted as filled black boxes, 3′UTRs as white boxes, introns as lines, large AGEZ (>100 nt) in thick gray lines. Known alternative splicing events are indicated by dashed lines. (D) Experimental strategy: Each exon with a large AGEZ was cloned along with upstream and downstream intronic sequences into the GFP-based exon trapping construct driven by chicken β-actin promoter (cBA) for tissue culture transfection. The AGEZ (in gray) with some additional upstream intronic region was cloned into the PY7 in vitro splicing construct to replace the BP, PPT, and 3′ ss elements of TPM1 exon 3 (TM3).