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

Abstract

Alternative pre-mRNA splicing is a major gene expression regulatory mechanism in metazoan organisms. Proteins that bind pre-mRNA elements and control assembly of splicing complexes regulate utilization of pre-mRNA alternative splice sites. To understand how signaling pathways impact this mechanism, an RNA interference screen in Drosophila S2 cells was used to identify proteins that regulate TAF1 (TBP-associated factor 1) alternative splicing in response to activation of the ATR (ATM-RAD3-related) signaling pathway by the chemotherapeutic drug camptothecin (CPT). The screen identified 15 proteins that, when knocked down, caused the same change in TAF1 alternative splicing as CPT treatment. However, combined RNA interference and CPT treatment experiments indicated that only a subset of the identified proteins are targets of the CPT-induced signal, suggesting that multiple independent pathways regulate TAF1 alternative splicing. To understand how signals modulate the function of splicing factors, we characterized one of the CPT targets, Tra2 (Transformer-2). CPT was found to downregulate Tra2 protein levels. CPT-induced Tra2 down-regulation was ATR-dependent and temporally paralleled the change in TAF1 alternative splicing, supporting the conclusion that Tra2 directly regulates TAF1 alternative splicing. Additionally, CPT-induced Tra2 down-regulation occurred independently of new protein synthesis, suggesting a post-translational mechanism. The proteasome inhibitor MG132 reduced CPT-induced Tra2 degradation and TAF1 alternative splicing, and mutation of evolutionarily conserved Tra2 lysine 81, a potential ubiquitin conjugation site, to arginine inhibited CPT-induced Tra2 degradation, supporting a proteasome-dependent alternative splicing mechanism. We conclude that CPT-induced TAF1 alternative splicing occurs through ATR-signaled degradation of a subset of splicing-regulatory proteins.Alternative splicing is a fundamental mechanism operating in metazoan organisms to regulate protein expression (1-3). In humans and Drosophila, 30 -95% of pre-mRNAs undergo alternative splicing, resulting in mature mRNAs that encode proteins with different sequences or that contain a premature stop codon and are subject to degradation (4 -11). Alternative splicing plays key roles in developmental processes, such as Drosophila sex determination, and defects in alternative splicing are commonly associated with human disorders, such as spinal muscular atrophy (12, 13). Thus, understanding the molecular details of alternative splicing mechanisms has global implications for understanding both normal and disease states.At its basic level, the choice of splicing pattern for a pre-mRNA involves the regulation of spliceosome assembly on introns to be excised. Spliceosomes contain five small nuclear ribonucleoproteins (snRNPs) 3 (U1, U2, U4, U5, and U6) and numerous regulatory proteins, including the heterodimeric complex U2AF (U2 auxiliary factor) (14 -16). Spliceosomes serve to define 5Ј and 3Ј splice sites at exon/intron boundaries and carry...

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

“…Among these candidates, the Half-pint (Hfp) protein, also known as Puf68, was selected for further analysis as it has previously been implicated in the regulation of alternative splicing. 30,32 the protein binds relative to the affected splice site. [21][22][23][24] In the studies of Tra2, it was observed that Tra2 bound at intronic positions elicits splicing repression, but binding at exonic positions causes splicing activation.…”

“…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.…”

Hippo signaling promotes JNK-dependent cell migration

“…5A). Rox8 encodes a RNA-binding protein that controls important aspects of development, including alternative splicing and stress granule formation (39,40). In addition, we have previously performed an unbiased genetic screen for factors modulating JNK signaling (41), and identified Rox8 as a positive regulator of JNK signaling for Rox8 expression synergistically enhances Egr-induced JNK-dependent cell death (Fig.…”

The cardiotonic steroid digitoxin regulates alternative splicing through depletion of the splicing factors SRSF3 and TRA2B

“…SR proteins can also repress alternative splicing through multiple mechanisms, including acting through intronic splicing silencer elements (ISSs) adjacent to an alternative exon (Kanopka et al 1996;Buratti et al 2007). Modulation of SR proteins by small molecules has been shown in some studies, and they present interesting therapeutic targets for misregulated alternative splicing events (Soret et al 2005;Katzenberger et al 2009). …”