Control of alternative splicing by signal-dependent degradation of splicing-regulatory proteins.
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 down-regulate 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.
Katzenberger
Rebeccah J
RJ
Department of Pharmacology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, USA.
Marengo
Matthew S
MS
Wassarman
David A
DA
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000166:cytology / Q000235:genetics; D005786:Gene Expression Regulation; D051548:Histone Acetyltransferases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D022821:RNA Splice Sites; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D020033:Protein Isoforms; D012322:RNA Precursors; D022821:RNA Splice Sites; D012261:Ribonucleoproteins; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; C066174:tra2 protein, Drosophila; D051548:Histone Acetyltransferases; C444435:Mei-41 protein, Drosophila; D017346:Protein Serine-Threonine Kinases; C069001:TATA-binding protein associated factor 250 kDa