Evidence for the function of an exonic splicing enhancer after the first catalytic step of pre-mRNA splicing.
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.
Chew
S L
SL
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
Liu
H X
HX
Mayeda
A
A
Krainer
A R
AR
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000256:Adenoviridae / Q000235:genetics; D002384:Catalysis; D004742:Enhancer Elements, Genetic / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D007075:Immunoglobulin M / Q000378:metabolism; D007700:Kinetics; D008957:Models, Genetic; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing / Q000502:physiology*; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D013997:Time Factors; D020040:Trans-Splicing / Q000502:physiology
D007075:Immunoglobulin M; D009687:Nuclear Proteins; D009841:Oligonucleotides; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D000255:Adenosine Triphosphate