An interaction between U2AF 65 and CF Im links the splicing and 3′ end processing machineries

Millevoi, Stefania; Loulergue, Clarisse; Dettwiler, Sabine; Karaa, Sarah Zeïneb; Keller, Walter; Antoniou, Michael; Vagner, Stéphan

doi:10.1038/sj.emboj.7601331

Cited by 180 publications

(197 citation statements)

References 46 publications

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“…Therefore, it is tempting to speculate that a fine-tuning regulation is operating to control the amount of each factor to compensate the cellular balance of free U2AF65 or U2AF35 and U2AF65-U2AF35 heterodimer. Additionally, U2AF65 has been implicated in the regulation of 3Ј end processing (Millevoi et al, 2006;Danckwardt et al, 2007). In this regard, we speculate that the coupling of splicing and 3Ј end processing of mammalian pre-mRNAs should be altered as well.…”

Section: Cellular Consequences Of U2af65 Cleavage By Caspasesmentioning

confidence: 87%

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

Izquierdo

2008

MBoC

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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...

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Section: Cellular Consequences Of U2af65 Cleavage By Caspasesmentioning

confidence: 87%

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

Izquierdo

2008

MBoC

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“…First and last exons only have one flanking splice site and are, therefore, recognized differently than internal exons. Terminal exon definition has been shown to be aided by the polyadenylation machinery 14,15 through interactions between U2AF and the polyadenylation polymerase (PAP) 16 or cleavage factor 1m (CF1m), 17 or through interactions between splicing factor 3b (SF3b), a component of U2 snRNP, and the cleavage and polyadenylation specificity factor (CPSF). 18 Furthermore, the U1 snRNP component U1A has been shown to stimulate polyadenylation through interaction with CPSF160, 19 however, U1 snRNP also plays a role in preventing premature cleavage and polyadenylation through binding of U1 snRNA to cryptic poly(A) sites 20 as well as through direct interactions between U1-70K and the PAP.…”

Section: Introductionmentioning

confidence: 99%

Coupling between alternative polyadenylation and alternative splicing is limited to terminal introns

et al. 2016

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Alternative polyadenylation has been implicated as an important regulator of gene expression. In some cases, alternative polyadenylation is known to couple with alternative splicing to influence last intron removal. However, it is unknown whether alternative polyadenylation events influence alternative splicing decisions at upstream exons. Knockdown of the polyadenylation factors CFIm25 or CstF64 in HeLa cells was used as an approach in identifying alternative polyadenylation and alternative splicing events on a genome-wide scale. Although hundreds of alternative splicing events were found to be differentially spliced in the knockdown of CstF64, genes associated with alternative polyadenylation did not exhibit an increased incidence of alternative splicing. These results demonstrate that the coupling between alternative polyadenylation and alternative splicing is usually limited to defining the last exon. The striking influence of CstF64 knockdown on alternative splicing can be explained through its effects on UTR selection of known splicing regulators such as hnRNP A2/B1, thereby indirectly influencing splice site selection. We conclude that changes in the expression of the polyadenylation factor CstF64 influences alternative splicing through indirect effects.

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“…As a class, they are expected to have similar structures and active sites (Stern et al 2007) and would therefore be expected to share sensitivity to the same inhibitors. While several of these paralogs are known to localize to the nucleus (Moorhead et al 2007), one in particular, PP2Cg (PPM1G), has been found to play a role in pre-mRNA splicing (Murray et al 1999;Allemand et al 2007), which is in turn coordinated with 39 cleavage (Niwa et al 1990;Kyburz et al 2006;Millevoi et al 2006). We detected PP2Cg at varying levels by Western blotting in our partially purified cleavage factors (Fig.…”

Section: Resultsmentioning

confidence: 99%

Small molecule activators of pre-mRNA 3′ cleavage

Ryan

Khleborodova²,

Pan³

et al. 2009

RNA

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39 Cleavage and polyadenylation are obligatory steps in the biogenesis of most mammalian pre-mRNAs. In vitro reconstitution of the 39 cleavage reaction from human cleavage factors requires high concentrations of creatine phosphate (CP), though how CP activates cleavage is not known. Previously, we proposed that CP might work by competitively inhibiting a cleavagesuppressing serine/threonine (S/T) phosphatase. Here we show that fluoride/EDTA, a general S/T phosphatase inhibitor, activates in vitro cleavage in place of CP. Subsequent testing of inhibitors specific for different S/T phosphatases showed that inhibitors of the PPM family of S/T phosphatases, which includes PP2C, but not the PPP family, which includes PP1, PP2A, and PP2B, activated 39 cleavage in vitro. In particular, NCI 83633, an inhibitor of PP2C, activated extensive 39 cleavage at a concentration 50-fold below that required by fluoride or CP. The testing of structural analogs led to the identification of a more potent compound that activated 39 cleavage at 200 mM. While testing CP analogs to understand the origin of its cleavage activation effect, we found phosphocholine to be a more effective activator than CP. The minimal structural determinants of 39 cleavage activation by phosphocholine were identified. Our results describe a much improved small molecule activator of in vitro pre-mRNA cleavage, identify the molecular determinants of cleavage activation by phosphoamines such as phosphocholine, and suggest that a PPM family phosphatase is involved in the negative regulation of mammalian pre-mRNA 39 cleavage.

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An interaction between U2AF 65 and CF Im links the splicing and 3′ end processing machineries

Cited by 180 publications

References 46 publications

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

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

Coupling between alternative polyadenylation and alternative splicing is limited to terminal introns

Small molecule activators of pre-mRNA 3′ cleavage

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