Pre-mRNA splicing of IgM exons M1 and M2 is directed by a juxtaposed splicing enhancer and inhibitor.
Splicing of certain pre-mRNA introns is dependent on an enhancer element, which is typically purine-rich. It is generally thought that enhancers increase the use of suboptimal splicing signals, and one specific proposal is that enhancers stabilize binding of U2AF65 to weak polypyrimidine (Py) tracts. Here, we test this model using an IgM pre-mRNA substrate, which contains a well-characterized enhancer. Although the enhancer was required for in vitro splicing, we found it had no effect on U2AF65 binding. Unexpectedly, replacement of the natural IgM Py tract, branchpoint, and 5' splice site with consensus splicing signals did not circumvent the enhancer requirement. These observations led us to identify a novel regulatory element within the IgM M2 exon that acts as a splicing inhibitor; removal of the inhibitor enabled splicing to occur in the absence of the enhancer. The IgM M2 splicing inhibitor is evolutionarily conserved, can inhibit the activity of an unrelated, constitutively spliced pre-mRNA, and acts by repressing splicing complex assembly. Interestingly, the inhibitor itself forms an ATP-dependent complex that contains U2 snRNP. We conclude that splicing of IgM exons M1 and M2 is directed by two juxtaposed regulatory elements-an enhancer and an inhibitor-and that a primary function of the enhancer is to counteract the inhibitor.
Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester, Massachusetts 01605, USA.
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Green
D001483:Base Sequence; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D005914:Globins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007075:Immunoglobulin M / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000235:genetics; D012261:Ribonucleoproteins / Q000235:genetics*; D017385:Sequence Homology; D000072668:Splicing Factor U2AF
D007075:Immunoglobulin M; D009687:Nuclear Proteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; D005914:Globins