A DNA damage signal activates and derepresses exon inclusion in Drosophila TAF1 alternative splicing

Abstract

Signal-dependent alternative splicing is important for regulating gene expression in eukaryotes, yet our understanding of how signals impact splicing mechanisms is limited. A model to address this issue is alternative splicing of Drosophila TAF1 pre-mRNA in response to camptothecin (CPT)-induced DNA damage signals. CPT treatment of Drosophila S2 cells causes increased inclusion of TAF1 alternative cassette exons 12a and 13a through an ATR signaling pathway. To evaluate the role of TAF1 premRNA sequences in the alternative splicing mechanism, we developed a TAF1 minigene (miniTAF1) and an S2 cell splicing assay that recapitulated key aspects of CPT-induced alternative splicing of endogenous TAF1. Analysis of miniTAF1 indicated that splice site strength underlies independent and distinct mechanisms that control exon 12a and 13a inclusion. Mutation of the exon 13a weak 59 splice site or weak 39 splice site to a consensus sequence was sufficient for constitutive exon 13a inclusion. In contrast, mutation of the exon 12a strong 59 splice site or moderate 39 splice site to a consensus sequence was only sufficient for constitutive exon 12a inclusion in the presence of CPT-induced signals. Analogous studies of the exon 13 39 splice site suggest that exon 12a inclusion involves signal-dependent pairing between constitutive and alternative splice sites. Finally, intronic elements identified by evolutionary conservation were necessary for full repression of exon 12a inclusion or full activation of exon 13a inclusion and may be targets of CPT-induced signals. In summary, this work defines the role of sequence elements in the regulation of TAF1 alternative splicing in response to a DNA damage signal.

Control of Alternative Splicing by Signal-dependent Degradation of Splicing-regulatory Proteins

“…The U2AF subunits U2AF38 and PUF60 may be integral to this mechanism, since alternative splicing can be controlled by signal-dependent regulation of U2AF occupancy on the pre-mRNA (57,60). Consistent with this scenario, mutating the exon 13 3Ј splice site to improve the match to the 3Ј splice site consensus sequence increases exclusion of exon 12a (26). In this scenario, the six CPT-targeted splicing-regulatory proteins may function coordinately to define exon 13 splice sites, explaining why loss of any one of the proteins by RNAi is sufficient to activate exon 12a and 13a inclusion.…”

“…Thus, in the case of TAF1 alternative splicing, Tra2 may bind intronic splicing silencers in introns flanking exons 12a and 13a to repress inclusion of these exons, and the CPT-induced reduction in Tra2 level relieves this repression. Although binding sites for Tra2 in the TAF1 pre-mRNA have not been identified, an evolutionarily conserved intronic splicing silencer, termed IE-A, is located in the intron between exons 12 and 12a (26).…”

“…In addition, as indicated by studies in which the strength of TAF1 splice sites was altered, up-regulation of TAF1-4 splicing involves altered recruitment of general splicing complexes to splice sites associated with exons 12a, 13, and 13a (26). Based on these findings, we hypothesized that splicing-regulatory proteins function downstream of the CPT-induced signaling pathway to mediate the change in TAF1 alternative splicing.…”

Half Pint/Puf68 is required for negative regulation of splicing by the SR factor Transformer2

“…32,40,41 These two factors were both among a small group of RNA binding proteins shown to be required to regulate splicing of mRNA encoding the Taf1-3 and Taf1-4 isoforms from the Drosophila Taf1 gene. The similar dependence of Taf1 on these two factors suggests that perhaps regulation occurs through a similar mechanism.…”

Comparative proteomics analysis of global cellular stress responses to hydroxyurea-induced DNA damage in HeLa cells

“…For example, one of down-regulated protein, programmed cell death protein 6 (PDCD6), also called apoptosislinked gene 2 protein (ALG2), functions as calciumbinding protein required for T cell receptor-, Fas-, and glucocorticoid-induced cell death (Suzuki et al 2012;Yoon et al 2012). Besides their multifunction, four proteins, ATP-dependent RNA helicase DDX39A, complement component 1 Q subcomponent-binding protein (SF2p32), Lamin-A/C, and far upstream element-binding Protein 2 are involved in pre-mRNA splicing or as splicing factors to play an important role in DNA damage and repair (Dittmer and Misteli 2011;Filippov et al 2007;Marengo and Wassarman 2008;Singh et al 2013). Replication factor C (RFC) subunit 5 is down regulated, suggesting that RFC and Pol d may be synergistically involved in DNA repair events on lagging or leading strand with their altered functions by the down-regulation of their smallest subunits.…”