Key result
A 3' exon containing the SF2/ASF-specific ESE underwent bimolecular exon ligation, whereas 3' exons without the ESE or with control sequences did not, suggesting ESEs can act late in splicing.
This basic science study demonstrates that exonic splicing enhancers can function late in the pre-mRNA splicing reaction to enhance the second catalytic step.
Extends basic splicing mechanisms in animal models; leaves open relevance to human cardiac transcript regulation.
Exonic splicing enhancers (ESEs) activate pre-mRNA splicing by promoting the use of the flanking splice sites. They are recognized by members of the serine/arginine-rich (SR) family of proteins, such as splicing factor 2/alternative splicing factor (SF2/ASF), which recruit basal splicing factors to form the initial complexes during spliceosome assembly. The in vitro splicing kinetics of an ESE-dependent IgM pre-mRNA suggested that an SF2/ASF-specific ESE has additional functions later in the splicing reaction, after the completion of the first catalytic step. A bimolecular exon ligation assay, which physically uncouples the first and second catalytic steps of splicing in a trans-splicing reaction, was adapted to test the function of the ESE after the first step. A 3' exon containing the SF2/ASF-specific ESE underwent bimolecular exon ligation, whereas 3' exons without the ESE or with control sequences did not. The ESE-dependent trans-splicing reaction occurred after inactivation of U1 or U2 small nuclear ribonucleoprotein particles, compatible with a functional assay for events after the first step of splicing. The ESE-dependent step appears to take place before the ATP-independent part of the second catalytic step. Bimolecular exon ligation also occurred in an S100 cytosolic extract, requiring both the SF2/ASF-dependent ESE and complementation with SF2/ASF. These data suggest that some ESEs can act late in the splicing reaction, together with appropriate SR proteins, to enhance the second catalytic step of splicing.
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Chew et al. (1999) studied this question. SF2/ASF-specific ESE vs. 3' exons without the ESE or with control sequences was evaluated on Bimolecular exon ligation (second catalytic step of splicing). A 3' exon containing the SF2/ASF-specific ESE underwent bimolecular exon ligation, whereas 3' exons without the ESE or with control sequences did not, suggesting ESEs can act late in splicing.
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