Phosphorylation-Dependent Regulation of PSF by GSK3 Controls CD45 Alternative Splicing

Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, 422 Curie Boulevard, Philadelphia, PA 19104-6059, USA.
Molecular cell (Impact Factor: 14.02). 10/2010; 40(1):126-37. DOI: 10.1016/j.molcel.2010.09.013
Source: PubMed


Signal-induced alternative splicing of the CD45 gene in human T cells is essential for proper immune function. Skipping of the CD45 variable exons is controlled, in large part, by the recruitment of PSF to the pre-mRNA substrate upon T cell activation; however, the signaling cascade leading to exon exclusion has remained elusive. Here we demonstrate that in resting T cells PSF is directly phosphorylated by GSK3, thus promoting interaction of PSF with TRAP150, which prevents PSF from binding CD45 pre-mRNA. Upon T cell activation, reduced GSK3 activity leads to reduced PSF phosphorylation, releasing PSF from TRAP150 and allowing it to bind CD45 splicing regulatory elements and repress exon inclusion. Our data place two players, GSK3 and TRAP150, in the complex network that regulates CD45 alternative splicing and demonstrate a paradigm for signal transduction from the cell surface to the RNA processing machinery through the multifunctional protein PSF.

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    • "More recent studies have demonstrated that Thrap3 is a selective transcriptional coactivator for CLOCK–BMAL1, a common circadian clock factor, and depletion of Thrap3 causes low-amplitude, long-period circadian rhythms, identifying it as a positive clock element (Lande-Diner et al. 2013). In addition, it has been reported that phosphorylation of PSF/SFPQ by glycogen synthase kinase 3 (GSK3) promotes an interaction with Thrap3 and prevents PSF from binding to the CD45 promoter (Heyd and Lynch 2010). In this model, the crucial regulatory idea is the phosphorylation-dependent interaction of PSF with Thrap3. "
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    Genes & Development 10/2014; 28(21). DOI:10.1101/gad.249367.114 · 10.80 Impact Factor
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    • "Triiodothyronine (T3) activates the thyroid hormone receptor (TR), which binds to the thyroid hormone-response element (TRE) in both the absence and presence of a ligand [17], and has previously been shown to modulate the AS of beta-amyloid and TRa genes expressed in cultured cells [18] [19]. The TR-associated protein, 150-kDa (TRAP150) (also known as TR-associated pro- tein3, THRAP3), was originally isolated as a subunit of the TRAP/ Mediator complex, which can be recruited to liganded TR and facilitate the recruitment of pol II to initiate transcription [20], and has recently been shown to play a role in pre-mRNA splicing [21] [22] [23]. "
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    ABSTRACT: Emerging evidence has indicated that the transcription and processing of precursor mRNA (pre-mRNA) are functionally coupled to modulate gene expression. In collaboration with coregulators, several steroid hormone receptors have previously been shown to directly affect alternative pre-mRNA splicing coupled to hormone-induced gene transcription; however, the roles of the thyroid hormone receptor (TR) and its coregulators in alternative splicing coordinated with transcription remain unknown. In the present study, we constructed a luciferase reporter and CD44 alternative splicing (AS) minigene driven by a minimal promoter carrying 2 copies of the palindromic thyroid hormone-response element. We then examined whether TR could modulate pre-mRNA processing coupled to triiodothyronine (T3)-induced gene transcription using luciferase reporter and splicing minigene assays in HeLa cells. In the presence of cotransfected TRβ1, T3 increased luciferase activities along with the inclusion of the CD44 variable exons 4 and 5 in a dose- and time-dependent manner. In contrast, cotransfected TRβ1 did not affect the exon-inclusion of the CD44 minigene driven by the cytomegalovirus promoter. T3-induced two-exon inclusion was significantly increased by the cotransfection of the TR-associated protein, 150-kDa, a subunit of the TRAP/Mediator complex that has recently been shown to function as a splicing factor. In contrast, T3-induced two-exon inclusion was significantly decreased by cotransfection of the polypyrimidine tract-binding protein-associated splicing factor, which was previously shown to function as a corepressor of TR. These results demonstrated that liganded TR in cooperation with its associating cofactors could modulate alternative pre-mRNA splicing coupled to gene transcription.
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    • "We then tested regulation of the endogenous Rac1b levels in NCM460 cells using depletion of either SRSF1 or SRSF3 by RNA interference and also observed the described antagonistic regulation (Fig. 1B). Once validated as a working model, these cells were transfected with a selection of shRNA plasmids from a previously described library (Moura-Alves et al. 2011; Moita et al. 2012), directly targeting a set of 20 kinases and phosphatases (see Table 1) earlier implicated in the regulation of other alternative splicing events (Blaustein et al. 2005; Ngo et al. 2005; Heyd and Lynch 2010; Liu et al. 2011; Qian et al. 2011). As a crude primary screening method, protein extracts from these cells were analyzed by Western blot for changes in endogenous Rac1b levels. "
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    ABSTRACT: The premessenger RNA of the majority of human genes can generate various transcripts through alternative splicing, and different tissues or disease states show specific patterns of splicing variants. These patterns depend on the relative concentrations of the splicing factors present in the cell nucleus, either as a consequence of their expression levels or of post-translational modifications, such as protein phosphorylation, which are determined by signal transduction pathways. Here, we analyzed the contribution of protein kinases to the regulation of alternative splicing variant Rac1b that is overexpressed in certain tumor types. In colorectal cells, we found that depletion of AKT2, AKT3, GSK3β, and SRPK1 significantly decreased endogenous Rac1b levels. Although knockdown of AKT2 and AKT3 affected only Rac1b protein levels suggesting a post-splicing effect, the depletion of GSK3β or SRPK1 decreased Rac1b alternative splicing, an effect mediated through changes in splicing factor SRSF1. In particular, the knockdown of SRPK1 or inhibition of its catalytic activity reduced phosphorylation and subsequent translocation of SRSF1 to the nucleus, limiting its availability to promote the inclusion of alternative exon 3b into the Rac1 pre-mRNA. Altogether, the data identify SRSF1 as a prime regulator of Rac1b expression in colorectal cells and provide further mechanistic insight into how the regulation of alternative splicing events by protein kinases can contribute to sustain tumor cell survival.
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