Abstract
BackgroundDuring spliceosome assembly, protein-protein interactions (PPI) are sequentially formed and disrupted to accommodate the spatial requirements of pre-mRNA substrate recognition and catalysis. Splicing activators and repressors, such as SR proteins and hnRNPs, modulate spliceosome assembly and regulate alternative splicing. However, it remains unclear how they differentially interact with the core spliceosome to perform their functions.ResultsHere, we investigate the protein connectivity of SR and hnRNP proteins to the core spliceosome using probabilistic network reconstruction based on the integration of interactome and gene expression data. We validate our model by immunoprecipitation and mass spectrometry of the prototypical splicing factors SRSF1 and hnRNPA1. Network analysis reveals that a factor’s properties as an activator or repressor can be predicted from its overall connectivity to the rest of the spliceosome. In addition, we discover and experimentally validate PPIs between the oncoprotein SRSF1 and members of the anti-tumor drug target SF3 complex. Our findings suggest that activators promote the formation of PPIs between spliceosomal sub-complexes, whereas repressors mostly operate through protein-RNA interactions.ConclusionsThis study demonstrates that combining in-silico modeling with biochemistry can significantly advance the understanding of structure and function relationships in the human spliceosome.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0682-5) contains supplementary material, which is available to authorized users.
Highlights
During spliceosome assembly, protein-protein interactions (PPI) are sequentially formed and disrupted to accommodate the spatial requirements of pre-mRNA substrate recognition and catalysis
Splicing efficiency and alternative splicing regulation are controlled by trans-acting splicing factors, which bind to cis-acting elements on the pre-mRNA to either activate or repress the selection of particular splice sites [6]
We discovered that SRSF1 forms multiple PPIs with the earlyacting U2-small nuclear ribonucleoprotein complexes (snRNPs)-specific SF3 complex, which we confirmed by in-vitro pull-down experiments
Summary
Protein-protein interactions (PPI) are sequentially formed and disrupted to accommodate the spatial requirements of pre-mRNA substrate recognition and catalysis. Splicing activators and repressors, such as SR proteins and hnRNPs, modulate spliceosome assembly and regulate alternative splicing It remains unclear how they differentially interact with the core spliceosome to perform their functions. The spliceosome forms step-wise on the pre-mRNA [2], through sequential rearrangements in which various protein and RNP complexes form and disassemble distinct proteinprotein interactions (PPIs), in addition to RNA-RNA and RNA-protein interactions. These transformations, The structural plasticity of the spliceosome makes it susceptible to regulation, allowing for the skipping or inclusion of alternative exons or exon segments [2], known as alternative splicing. Splicing efficiency and alternative splicing regulation are controlled by trans-acting splicing factors, which bind to cis-acting elements on the pre-mRNA to either activate or repress the selection of particular splice sites [6]
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