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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    • "Depletion of PSF was accomplished by lentivirus encoding hairpins targeted to PSF cDNA encoding residues 464–470 (AQKNPMY). Depletion of TRAP150 was performed by antisense morpholino knockdown as previously described (Heyd and Lynch, 2010). "
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    ABSTRACT: PSF (a.k.a. SFPQ) is a ubiquitously expressed, essential nuclear protein with important roles in DNA damage repair and RNA biogenesis. In stimulated T cells, PSF binds to and suppresses the inclusion of CD45 exon 4 in the final mRNA; however, in resting cells, TRAP150 binds PSF and prevents access to the CD45 RNA, though the mechanism for this inhibition has remained unclear. Here, we show that TRAP150 binds a region encompassing the RNA recognition motifs (RRMs) of PSF using a previously uncharacterized, 70 residue region we have termed the PSF-interacting domain (PID). TRAP150's PID directly inhibits the interaction of PSF RRMs with RNA, which is mediated through RRM2. However, interaction of PSF with TRAP150 does not appear to inhibit the dimerization of PSF with other Drosophila Behavior, Human Splicing (DBHS) proteins, which is also dependent on RRM2. Finally, we use RASL-Seq to identify ∼40 T cell splicing events sensitive to PSF knockdown, and show that for the majority of these, PSF's effect is antagonized by TRAP150. Together these data suggest a model in which TRAP150 interacts with dimeric PSF to block access of RNA to RRM2, thereby regulating the activity of PSF toward a broad set of splicing events in T cells. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
    Full-text · Article · Aug 2015 · Nucleic Acids Research
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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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    ABSTRACT: Phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ) at Ser273 by cyclin-dependent kinase 5 (CDK5) in adipose tissue stimulates insulin resistance, but the underlying molecular mechanisms are unclear. We show here that Thrap3 (thyroid hormone receptor-associated protein 3) can directly interact with PPARγ when it is phosphorylated at Ser273, and this interaction controls the diabetic gene programming mediated by the phosphorylation of PPARγ. Knockdown of Thrap3 restores most of the genes dysregulated by CDK5 action on PPARγ in cultured adipocytes. Importantly, reduced expression of Thrap3 in fat tissue by antisense oligonucleotides (ASOs) regulates a specific set of genes, including the key adipokines adiponectin and adipsin, and effectively improves hyperglycemia and insulin resistance in high-fat-fed mice without affecting body weight. These data indicate that Thrap3 plays a crucial role in controlling diabetic gene programming and may provide opportunities for the development of new therapeutics for obesity and type 2 diabetes.
    Preview · Article · Oct 2014 · Genes & Development
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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.
    Preview · Article · Aug 2014 · Biochemical and Biophysical Research Communications
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