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

Abstract

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. INTRODUCTIONU2 small nuclear ribonucleoprotein (snRNP) auxiliary factors (U2AF65-U2AF35) cooperate in 3Ј splice site (3Јss) recognition through direct interactions with the branch point and the branch-binding protein SF1/BBP as well as with the polypyrimidine tract (PT) and the conserved AG dinucleotide of the 3Јss (Valcárcel et al., 1996;Merendino et al., 1999;Wu et al., 1999; Zorio and Blumenthal, 1999;Soares et al., 2006). U2AF65 contains three RNA recognition motifs (RRMs) and an N-terminal region rich in basic residues (RS domain). RRMs 1 and 2 of U2AF65 display a canonical RRM-fold, bind RNA in vitro, and are implicated in highaffinity binding to the PT (Zamore et al., 1992;Banerjee et al., 2003;Sickmier et al., 2006). RRM3 is a noncanonical RRM and mediates the SF1 interaction (Selenko et al., 2003). Recognition of the PT by U2AF65 is key to splice site selection and spliceosome assembly for the major class of introns in higher eukaryotes. Both length and pyrimidine content of this sequence are critical determinants of the relative strength of competing 3Јss and therefore of differential 3Јss use as a major source of proteome diversity (Reed, 1989;Izquierdo and Valcárcel, 2006). Apoptosis is induced by many different stimuli, including growth factor withdrawal, chemotherapeutic compounds, membrane-bound death receptors, or DNA-damaging agents (Krammer, 2000;Adams, 2003). Whatever the initial signal, common characteristic cytological and molecular changes occur during apoptosis (Adams, 2003). Several signaling components and apoptosis pathways have been identified and elucidated at the molecular level (Adams, 2003). When apoptosis is induced, caspases, a family of aspartylsp...

The Splicing Factor FUBP1 Is Required for the Efficient Splicing of Oncogene MDM2 Pre-mRNA

“…Interestingly, we also observed the binding of damage-specific cleaved forms of some of these proteins, including FUBP1, PTBP1, and U2AF65 to RNA2 ⌬1 in cisplatinum-treated nuclear extracts (Fig. 3B and data not shown) (48,49).…”

Alternative splicing ofU2AF1reveals a shared repression mechanism for duplicated exons

“…This increase was found also for constructs lacking the U2AF35 RS domain (lanes 3–4). U2AF35 knockdown was associated with the enhanced degradation of U2AF65 (Supplementary Figure S15), possibly through caspase activation (78), which could explain the observed compensatory increase of U2AF2 mRNAs in depleted cells (12). Expression of U2AF35a and U2AF35b constructs was also increased upon cotransfection with wild-type U2AF65 plasmids into untreated cells ( cf .…”

“…Blots were successively incubated with antibodies against Xpress (exU2AF35), U2AF35 (enU2AF35), GFP and U2AF65 (enU2AF65 and degU2AF65) ( C ). A C-terminal degradation product of U2AF65 was described previously in Jurkat cells (78). U2AF expression following addition of lysosomal inhibitor NH 4 Cl and immunoblotting with the Xpress antibody ( D ).…”

Interactome for auxiliary splicing factor U2AF65 suggests diverse roles

“…This domain was also shown to target U2AF 65 to the nucleus and was sufficient to target a heterologous protein to nuclear speckles [13], which are sub-nuclear structures enriched for splicing-, transcription-, and 3’ end RNA-processing-factors [14]. Interestingly, U2AF 65 is cleaved near aspartate 128 (D 128) by caspases during early apoptosis and the liberated RS-containing N-terminal fragment translocates from nuclear speckles to nucleolus-like structures [15]. Thus, regions outside of this N-terminal fragment might also participate in sub-nuclear localization [15].…”