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,...

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Bibliographic Details
Published in:The Journal of biological chemistry 2009-04, Vol.284 (16), p.10737-10746
Main Authors: Katzenberger, Rebeccah J., Marengo, Matthew S., Wassarman, David A.
Format: Article
Language:English
Subjects:
Alternative Splicing
Amino Acid Sequence
Animals
Cell Cycle Proteins - genetics
Cell Cycle Proteins - metabolism
Drosophila
Drosophila melanogaster - cytology
Drosophila melanogaster - genetics
Drosophila Proteins - genetics
Drosophila Proteins - metabolism
Gene Expression Regulation
Histone Acetyltransferases - genetics
Histone Acetyltransferases - metabolism
Humans
Metazoa
Molecular Sequence Data
Protein Isoforms - genetics
Protein Isoforms - metabolism
Protein-Serine-Threonine Kinases - genetics
Protein-Serine-Threonine Kinases - metabolism
Ribonucleoproteins - genetics
Ribonucleoproteins - metabolism
RNA Interference
RNA Precursors - genetics
RNA Precursors - metabolism
RNA Splice Sites
Signal Transduction - physiology
TATA-Binding Protein Associated Factors
Transcription Factor TFIID - genetics
Transcription Factor TFIID - metabolism
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M809506200