Alternative Splicing of RNA Triplets Is Often Regulated and Accelerates Proteome Evolution
Figure 4
Specific intronic sequence features regulate NAGNAG splicing.
(A) Illustration of NAGNAG minigene constructs, designed to test the roles of the branch point to 3′ splice site distance and of the −4 base in NAGNAG splicing. A short segment of intronic sequence spanning the branch point to the 3′ splice site of the PTBP2 NAGNAG was cloned upstream of the IGF2BP1 exon. To confirm the importance of a pyrimidine at the −4 position for distal NAG use, the effects of all four nucleotides at the −4 position were tested. The branch point to 3′ splice site distance was varied by introducing nucleotides (underlined in orange) in constructs containing the PTBP2 branch point sequence, or by removing nucleotides (indicated by green dots) in constructs containing the IGF2BP1 branch point sequence. Locations of RT-PCR primers are indicated by arrows. (B) Proximal isoform expression increased dramatically after the introduction of a purine at the −4 position. Splicing was monitored after minigene transfection into HEK293T cells by RT-PCR. Mean and standard deviation of at least three independent transfections are shown. A representative gel is shown below (top and bottom bands represent proximal and distal isoforms, respectively). (C) As in (B), but varying the branch point to 3′ splice site distance in the context of the native nPTB branch point sequence. The distance was increased by insertion of four or seven nucleotides of sequence of varying purine/pyrimidine composition as shown in (A). (D) As in (C), but decreasing the branch point to 3′ splice site distance in the context of the exogenous IGF2BP1 BPS by deletion of three or six bases as shown in (A).
