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Vol. 19, Issue 8, 3299-3307, August 2008

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Fas Splicing Regulation during Early Apoptosis Is Linked to Caspase-mediated Cleavage of U2AF65

Departamento de Biología Molecular, Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, DP 28049, Madrid, Spain

Submitted November 8, 2007; Revised May 14, 2008; Accepted May 15, 2008
Monitoring Editor: A. Gregory Matera

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 acid¹²⁸ 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.

Address correspondence to: José M. Izquierdo (jmizquierdo{at}cbm.uam.es)

Abbreviations used: NLS, nuclear localization signal; RRM, RNA recognition motif; RS, arginine-serine; SF1/BBP, branch-point binding protein; U2AF65, U2 snRNP auxiliary factor 65 kDa; U2AF35, U2 snRNP auxiliary factor 35 kDa.