Fox-3 and PSF interact to activate neural cell-specific alternative splicing

Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892, USA.
Nucleic Acids Research (Impact Factor: 9.11). 12/2010; 39(8):3064-78. DOI: 10.1093/nar/gkq1221
Source: PubMed


Fox-1 family (Fox) proteins, which consist of Fox-1 (A2BP1), Fox-2 (Rbm9) and Fox-3 (NeuN) in mammals, bind to the RNA element UGCAUG and regulate alternative pre-mRNA splicing. However the mechanisms for Fox-regulated splicing are largely unknown. We analyzed the expression pattern of the three Fox proteins as well as neural cell-specific alternative splicing of a cassette exon N30 of nonmuscle myosin heavy chain (NMHC) II-B in the mouse central nervous system. Histological and biochemical analyses following fluorescence-activated cell sorting demonstrate a positive correlation of N30 inclusion and Fox-3 expression. Further, we identified polypyrimidine tract binding protein-associated splicing factor (PSF) as an interacting protein with Fox-3 by affinity-chromatography. In cultured cells, enhancement of N30 inclusion by Fox-3 depends on the presence of PSF. PSF enhances N30 inclusion in a UGCAUG-dependent manner, although it does not bind directly to this element. Fox-3 is recruited to the UGCAUG element downstream of N30 in the endogenous NMHC II-B transcript in a PSF-dependent manner. This study is the first to identify PSF as a coactivator of Fox proteins and provides evidence that the Fox-3 and PSF interaction is an integral part of the mechanism by which Fox proteins regulate activation of alternative exons via a downstream intronic enhancer.

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    • "The expression vectors of human TRb1 (pKCR 2 -hTRb1), TRAP150 (pSV-SPORT-hTRAP150), and PSF (pCS3+MT-hPSF) were described previously [29] [30] [31]. The pGL4.23[luc2/minP] vector containing firefly luciferase (Luc2) cDNA under the control of a minimal promoter containing a TATAA box was obtained from Promega Corporation (Madison, MI). "
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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.
    Biochemical and Biophysical Research Communications 08/2014; 451(1). DOI:10.1016/j.bbrc.2014.07.029 · 2.30 Impact Factor
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    • "The Fox family of alternative splicing regulators contains three paralogs: Fox1 (also called A2BP1), Fox2 (also called RBM9) and Fox3 (also called HRNBP3 and NeuN). Fox2 is widely expressed in tissues, but Fox1 and Fox3 are most highly expressed in the brain (Kim et al., 2011; Nakahata and Kawamoto, 2005; Underwood et al., 2005). These proteins act as alternative splicing regulators and have been shown to act by binding to the hexanucleotide motif UGCAUG (Kim et al., 2009; Underwood et al., 2005), but can also recognize GCAUGU, GUGAUG, UGGUGA and GGUGGU (Yeo et al., 2009). "
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    ABSTRACT: We describe the discovery of sno-lncRNAs, a class of nuclear-enriched intron-derived long noncoding RNAs (lncRNAs) that are processed on both ends by the snoRNA machinery. During exonucleolytic trimming, the sequences between the snoRNAs are not degraded, leading to the accumulation of lncRNAs flanked by snoRNA sequences but lacking 5' caps and 3' poly(A) tails. Such RNAs are widely expressed in cells and tissues and can be produced by either box C/D or box H/ACA snoRNAs. Importantly, the genomic region encoding one abundant class of sno-lncRNAs (15q11-q13) is specifically deleted in Prader-Willi Syndrome (PWS). The PWS region sno-lncRNAs do not colocalize with nucleoli or Cajal bodies, but rather accumulate near their sites of synthesis. These sno-lncRNAs associate strongly with Fox family splicing regulators and alter patterns of splicing. These results thus implicate a previously unannotated class of lncRNAs in the molecular pathogenesis of PWS.
    Molecular cell 09/2012; 48(2). DOI:10.1016/j.molcel.2012.07.033 · 14.02 Impact Factor
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    • "Splicing regulatory proteins cannot only antagonize each other, but can also enhance their action on an exon, where they act like coactivators. This is exemplified by Fox-3 and PSF that together promote usage of a neuron-specific exon in the myosin heavy chain (Kim et al., 2011). "
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    ABSTRACT: Almost all polymerase II transcripts undergo alternative pre-mRNA splicing. Here, we review the functions of alternative splicing events that have been experimentally determined. The overall function of alternative splicing is to increase the diversity of mRNAs expressed from the genome. Alternative splicing changes proteins encoded by mRNAs, which has profound functional effects. Experimental analysis of these protein isoforms showed that alternative splicing regulates binding between proteins, between proteins and nucleic acids as well as between proteins and membranes. Alternative splicing regulates the localization of proteins, their enzymatic properties and their interaction with ligands. In most cases, changes caused by individual splicing isoforms are small. However, cells typically coordinate numerous changes in ‘splicing programs’, which can have strong effects on cell proliferation, cell survival and properties of the nervous system. Due to its widespread usage and molecular versatility, alternative splicing emerges as a central element in gene regulation that interferes with almost every biological function analyzed.
    Gene 08/2012; 514(1). DOI:10.1016/j.gene.2012.07.083 · 2.14 Impact Factor
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