Caspase-2 pre-mRNA alternative splicing: Identification of an intronic element containing a decoy 3' acceptor site.
We have established a model system using the caspase-2 pre-mRNA and initiated a study on the role of alternative splicing in regulation of programmed cell death. A caspase-2 minigene construct has been made that can be alternatively spliced in transfected cells and in nuclear extracts. Using this system, we have identified a 100-nt region in downstream intron 9 that inhibits the inclusion of the 61-bp alternative exon. This element (In100) can facilitate exon skipping in the context of competing 3' or 5' splice sites, but not in single-intron splicing units. The In100 element is also active in certain heterologous pre-mRNAs, although in a highly context-dependent manner. Interestingly, we found that In100 contains a sequence that highly resembles a bona fide 3' splice site. We provide evidence that this sequence acts as a "decoy" acceptor site that engages in U2 snRNP-dependent but nonproductive splicing complexes with the 5' splice site of exon 9, hence conferring competitive advantage to the exon-skipping splicing event (E8-E10). These results reveal a mechanism of action for a negative intronic regulatory element and uncover a role for U2 snRNP in the regulation of alternative splicing.
Coté
J
J
Department of Pediatrics and Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Dupuis
S
S
Jiang
Z
Z
Wu
J Y
JY
D020413:3' Untranslated Regions / Q000235:genetics; D017398:Alternative Splicing; D017209:Apoptosis / Q000502:physiology; D001483:Base Sequence; D053143:Caspase 2; D020169:Caspases / Q000235:genetics*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D005091:Exons; D005813:Genes, Synthetic; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D020413:3' Untranslated Regions; D012322:RNA Precursors; D011994:Recombinant Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D017413:Ribonucleoprotein, U2 Small Nuclear; D053143:Caspase 2; D020169:Caspases