Fas splicing regulation during early apoptosis is linked to caspase-mediated cleavage of U2AF65.
U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 kDa (U2AF65) is an essential splicing factor in the recognition of the pre-mRNA 3' splice sites during the assembly of the splicing commitment complex. We report here that U2AF65 is proteolyzed during apoptosis. This cleavage is group I or III caspase dependent in a noncanonical single site localized around the aspartic acid(128) residue and leads to the separation of the N- and C-terminal parts of U2AF65. The U2AF65 N-terminal fragment mainly accumulates in the nucleus within nuclear bodies (nucleoli-like pattern) and to a much lesser extent in the cytoplasm, whereas the C-terminal fragment is found in the cytoplasm, even in localization studies on apoptosis induction. From a functional viewpoint, the N-terminal fragment promotes Fas exon 6 skipping from a reporter minigene, by acting as a dominant-negative version of U2AF65, whereas the C-terminal fragment has no significant effect. The dominant-negative behavior of the U2AF65 N-terminal fragment can be reverted by U2AF35 overexpression. Interestingly, U2AF65 proteolysis in Jurkat cells on induction of early apoptosis correlates with the down-regulation of endogenous Fas exon 6 inclusion. Thus, these results support a functional link among apoptosis induction, U2AF65 cleavage, and the regulation of Fas alternative splicing.
Izquierdo
José M
JM
Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, DP 28049, Madrid, Spain. jmizquierdo@cbm.uam.es
D017398:Alternative Splicing; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D005091:Exons; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009687:Nuclear Proteins / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D012261:Ribonucleoproteins / Q000737:chemistry*; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry; D000072668:Splicing Factor U2AF; D019014:fas Receptor / Q000737:chemistry / Q000378:metabolism*
D009687:Nuclear Proteins; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; D019014:fas Receptor; D020169:Caspases