Homologues of the Caenorhabditis elegans Fox-1 Protein Are Neuronal Splicing Regulators in Mammals

Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095-1662, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 12/2005; 25(22):10005-16. DOI: 10.1128/MCB.25.22.10005-10016.2005
Source: PubMed


A vertebrate homologue of the Fox-1 protein from C. elegans was recently shown to bind to the element GCAUG and to act as an inhibitor of alternative splicing patterns in muscle. The
element UGCAUG is a splicing enhancer element found downstream of numerous neuron-specific exons. We show here that mouse
Fox-1 (mFox-1) and another homologue, Fox-2, are both specifically expressed in neurons in addition to muscle and heart. The
mammalian Fox genes are very complex transcription units that generate transcripts from multiple promoters and with multiple
internal exons whose inclusion is regulated. These genes produce a large family of proteins with variable N and C termini
and internal deletions. We show that the overexpression of both Fox-1 and Fox-2 isoforms specifically activates splicing of
neuronally regulated exons. This splicing activation requires UGCAUG enhancer elements. Conversely, RNA interference-mediated
knockdown of Fox protein expression inhibits splicing of UGCAUG-dependent exons. These experiments show that this large family
of proteins regulates splicing in the nervous system. They do this through a splicing enhancer function, in addition to their
apparent negative effects on splicing in vertebrate muscle and in worms.

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Available from: Joseph Dougherty, Feb 03, 2014
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    • "As shown by several groups, this sequence, when located in introns downstream of alternative exons, functions as a splicing enhancer to induce exon inclusion. It is suggested that intronic (U)GCAUG sequences upstream of regulated exons generally repress exon inclusion (Underwood et al., 2005), although this locational relationship may not be so strong (Zhang et al., 2008). Nevertheless, an upstream intronic (U)GCAUG sequence causes skipping of the alternative exon, F1c, and blocks formation of the prespliceosomal early complex (E-complex) (Fukumura et al., 2007). "
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    ABSTRACT: RBFox proteins are well-known alternative splicing regulators. We have shown previously that during neuronal differentiation of P19 cells induced by all-trans retinoic acid and cell aggregation, RBFox1 shows markedly increased temporal expression. To find its key splicing regulation, we examined the effect of RBFox1 on 33 previously reported and validated neuronal splicing events of P19 cells. We observed that alternative splicing of three genes, specifically, amyloid precursor protein (APP), disks large homolog 3 (DLG3), and G protein, alpha activating activity polypeptide O (GNAO1), was altered by transient RBFox1 expression in HEK293 and HeLa cells. Moreover, an RBFox1 mutant (RBFox1FA) that was unable to bind the target RNA sequence ((U)GCAUG) did not induce these splicing events. APP generates amyloid beta peptides that are involved in the pathology of Alzheimer’s disease, and therefore we examined APP alternative splicing regulation by RBFox1 and other splicing regulators. Our results indicated that RBFox proteins promote the skipping of APP exon 7, but not the inclusion of exon 8. We made APP6789 minigenes and observed that two (U)GCAUG sequences, located upstream of exon 7 and in exon 7, functioned to induce skipping of exon 7 by RBFox proteins. Overall, RBFox proteins may shift APP from exon 7 containing isoforms, APP770 and APP751, toward the exon 7 lacking isoform, APP695, which is predominant in neural tissues.
    Neurochemistry International 12/2014; 78. DOI:10.1016/j.neuint.2014.08.001 · 3.09 Impact Factor
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    • "In all cases, a skipping event produces a variant that lacks a portion of the RNA recognition motif (RRM) domain involved in recognizing the regulatory sequence (U/A)GCAUG [50]–[54]. RBFOX1 is specifically expressed in neurons, heart and muscle [52], [55]. RBFOX proteins are important regulators of muscle function in zebrafish where their depletion affects myofiber development [56]. "
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    ABSTRACT: With the goal of identifying splicing alterations in myotonic dystrophy 1 (DM1) tissues that may yield insights into targets or mechanisms, we have surveyed mis-splicing events in three systems using a RT-PCR screening and validation platform. First, a transgenic mouse model expressing CUG-repeats identified splicing alterations shared with other mouse models of DM1. Second, using cell cultures from human embryonic muscle, we noted that DM1-associated splicing alterations were significantly enriched in cytoskeleton (e.g. SORBS1, TACC2, TTN, ACTN1 and DMD) and channel (e.g. KCND3 and TRPM4) genes. Third, of the splicing alterations occurring in adult DM1 tissues, one produced a dominant negative variant of the splicing regulator RBFOX1. Notably, half of the splicing events controlled by MBNL1 were co-regulated by RBFOX1, and several events in this category were mis-spliced in DM1 tissues. Our results suggest that reduced RBFOX1 activity in DM1 tissues may amplify several of the splicing alterations caused by the deficiency in MBNL1.
    PLoS ONE 09/2014; 9(9):e107324. DOI:10.1371/journal.pone.0107324 · 3.23 Impact Factor
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    • "Additionally, the roles of motif spacing and of intervening sequences between tandem motifs remain largely uncharacterized. Widely used methods for mapping protein-RNA interactions in vivo based on ultraviolet cross-linking and immunoprecipitation (CLIP) (Ule et al., 2003; Underwood et al., 2005) have contributed to understanding of posttranscriptional regulation. However, these techniques are laborious and require many selection steps that likely introduce various types of bias. "
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    ABSTRACT: Specific protein-RNA interactions guide posttranscriptional gene regulation. Here, we describe RNA Bind-n-Seq (RBNS), a method that comprehensively characterizes sequence and structural specificity of RNA binding proteins (RBPs), and its application to the developmental alternative splicing factors RBFOX2, CELF1/CUGBP1, and MBNL1. For each factor, we recovered both canonical motifs and additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in motifs containing UGC and/or GCU. Dissociation constants calculated from RBNS data using a novel algorithm correlated highly with values measured by surface plasmon resonance. Motifs identified by RBNS were conserved, were bound and active in vivo, and distinguished the subset of motifs enriched by CLIP-Seq that had regulatory activity. Together, our data demonstrate that RBNS complements crosslinking-based methods and show that in vivo binding and activity of these splicing factors is driven largely by intrinsic RNA affinity.
    Molecular cell 05/2014; 54(5). DOI:10.1016/j.molcel.2014.04.016 · 14.02 Impact Factor
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