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CELF1, a Multifunctional Regulator of Posttranscriptional Networks

Written By

Daniel Beisang, Paul R. Bohjanen and Irina A. Vlasova-St. Louis

Submitted: 12 January 2012 Published: 19 September 2012

DOI: 10.5772/48780

From the Edited Volume

Binding Protein

Edited by Kotb Abdelmohsen

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1. Introduction

In order to assure the precise utilization of genetic information, gene expression is regulated at the level of transcription as well as multiple post-transcriptional levels including splicing, transport, localization, mRNA stability, and translation [1],[2],[3],[4],[5],[6],[7]. During evolution, cells developed precise mechanisms to ensure that each transcript is appropriately stored, modified, translated or degraded, depending on the need for the mRNA or encoded protein by the cell. Steady-state protein levels within a cell correlate poorly with steady-state levels of mRNA, leading scientists to hypothesize that the gene expression is regulated at post-transcriptional levels [8]. Work over the past quarter century has resulted in the identification of unifying concepts in post-transcriptional regulation. One unifying concept states is that post-transcriptional regulation is mediated by two major molecular components: cis-acting regulatory sequence elements and trans-acting factors. Cis-acting regulatory sequence elements are sub-sequences contained in the 5’ untranslated region (UTR), coding region, and 3’UTR of mRNA that are selectively recognized by a complementary set of one or more trans-acting factors to regulate post-transcriptional gene expression. Trans-actingfactors include RNA-binding proteins (RBPs) and microRNAs (miRNAs) which are able to influence the fate of mRNA by controlling processes such as translation and mRNA degradation (reviewed in references [9],[10],[11],[12]). The combinatorial interplay between various miRNAs and RBPs binding to a given mRNA allows for the transcript specific regulation critical to many cellular decisions during development [13],[14],[15],[16] and in response to environmental stimuli (reviewed in references [17],[18],[19],[20],[21],[22]).

Various experimental approaches have been developed to understand the interaction between RBPs and the network of transcripts that they regulate. One of the most widely used techniques involves immunopurification of specific RNA-binding proteins from cellular extracts followed by high-throughput analysis of the co-purified RNA species [23]. The coupling of this technique to powerful bioinformatic analysis methods has lead researchers to understand the binding specificity of a wide-variety of RBPs. The advent of new technology such as next generation sequencing and chemical cross-linking procedures have improved these methodologies and allowed for the fine-scale mapping of RBP binding sites, as well as the refinement of RBP binding motifs. Microarray-based studies that evaluated mRNA decay rates on a global basis have also provided valuable information about the role of post-transcriptional regulation of a wide variety of transcripts that have important physiological functions [24],[25],[26],[27],[28],[29].

This chapter focuses on the role of CELF1 (CUGBP and embryonically lethal abnormal vision-type RNA binding protein 3-like factor 1) in the regulation of posttranscriptional gene expression. CELF1 functions to regulate posttranscriptional gene expression by binding to RNA sequences known as GU-rich elements (GREs). Genome-wide measurements of mRNA decay and bioinformatic sequence motif discovery methods were used to identify the GRE as a highly conserved sequence that was enriched in the 3’UTR of mRNA transcripts with short half lives in primary human T lymphocytes [30]. This sequence resembled previously characterized binding sites for CELF1 [31],[32], and CELF1 was found to bind with high affinity to GRE sequences and mediate mRNA degradation [30]. This chapter reviews how CELF1 and its target transcripts function as an evolutionarily conserved posttranscriptional regulatory network which plays important roles in health and disease.

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2. Evolutionary conservation of CELF proteins

The CELF protein family is an evolutionarily conserved family of RNA-binding proteins that play essential roles in post-transcriptional gene regulation [28],[33]. These proteins contain three highly conserved RNA-Recognition Motifs (RRM) with the 2 N-terminal RRMs and the C-terminal RRM being separated by a highly divergent linker domain [34]. The RRMs confer RNA binding activity, and it is postulated that the divergent linker domain is an important site for functional regulation. Six members of the CELF family have been identified in humans and mice: CELF1 (CUGBP1) and CELF2 (CUGBP2) proteins are expressed ubiquitously and play vital role in embryogenesis [35],[36],[37],[38],[39], whereas CELF proteins 3-6 are restricted to adult tissues and found almost exclusively in the nervous system [40],[41]. CELF proteins often serve multiple functions in both the cytoplasm and the nucleus [42],[43]. Human CELF1 and its orthologs in Gallus gallus, Zebrafish, Xenopus, Drosophila and C. elegans have been known for many years to regulate gene expression at posttranscriptional levels and to control important developmental processes [31],[44],[45],[46],[47],[48],[49].

CELF1 function is conserved across evolution at the level of biochemical mechanism as well as its function in regulating development. Transcript deadenylation is often the first step in the mRNA degradation process, and CELF1 has been shown to promote transcript deadenylation in diverse species [28],[50]. In Xenopus embryos the CELF1 homologue Embryo Deadenylation Element Binding Protein (EDEN-BP), which is 88% identical to CELF1, regulates transcript deadenylation, and human CELF1 was able to functionally replace the deadenylation function of EDEN-BP in Xenopus extracts [51]. In HeLa cell extracts CELF1 also promotes transcript deadenylation and was shown to recruit PolyA Ribonuclease (PARN) [52]. In addition to the evolutionary conservation of the biochemical function of CELF1, the developmental programs regulated by CELF1 may also be conserved. For example, CELF1 appears to be an important factor in muscle development in diverse species. Studies investigating the function of CELF1 in Drosophila, Xenopus and mice have shown that CELF1 is critical for regulating the muscle developmental program [45], [53], [54]. More recent work suggests that CELF1 is a crucial factor in the regulation of mRNA degradation in mouse myoblasts [55]. Thus, in addition to the conservation of its biochemical function as a regulator of deadenylation, the role of CELF1 in muscle development may also be conserved.

As described below, CELF proteins from diverse species bind to RNA preferentially at GU-rich sequences and thereby regulate post-transcriptional processes such as mRNA splicing, translation, deadenylation and mRNA degradation. The structure and biochemical properties of CELF family members suggest functional redundancy [56], yet each CELF protein targets specific sub-populations of RNA transcripts and appears to have distinct functions [57]. We are starting to understand the mechanisms by which an individual CELF protein can serve multiple biochemical functions to coordinately regulate gene expression at posttranscriptional levels [30].

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3. Biochemistry of binding by CELF proteins to target mRNA

CELF 1 and 2 proteins were first isolated and characterized as novel heterogeneous nuclear ribonucleoproteins (hnRNPs). Timchenko et.al. demonstrated that these proteins bound to RNA containing the sequence (CUG)8 within the 3'UTR of myotonin protein kinase mRNA in vitro[58],[59]. Subsequent searches for the RNA-binding specificities of CELF1 and CELF2 used systemic evolution of ligands exponential enrichment (SELEX), revealing that CELF1 and CELF2 both bound preferentially to GU-rich RNA sequences [60]. Binding by CELF1 to GU-rich sequences in vitro and in vivo was abrogated by mutation of G nucleotides to C [30],[61]. Takanashi et. al. used a yeast three hybrid system for evaluating RNA-protein interactions, and found CELF1 bound preferentially to UG repeats rather than to CUG repeats [32]. CELF1 bound with high specificity to (UG)15 based on a surface plasmon resonance (SPR) quantitative binding assay [62]. Orthologues of CELF1 in other species also appear to have preferences for binding to GU-rich sequences. In Xenopus, the CELF1 orthologue EDEN-BP (embryo deadenylation element binding protein) binds to the GU-rich EDEN element, which contains the sequence (UGUA)12, and functions as a deadenylation signal in Xenopus embryos after fertilization [31],[51]. In Drosophila, the CELF1 orthologue Bru-3 was found to bind specifically to (UG)15 repeats and also was able to bind to the Xenopus EDEN element [46]. The Zebrafish protein Brul, a homologue of EDEN-BP with 81% identity, was also shown to preferentially bind to GU-rich RNAs [63]. EDEN-BP and Bru-3 can bind to GRE-RNA as dimers [64],[65] and may require GU-rich sequences of sufficient length to allow dimer formation [66]. In addition to the primary GU-rich sequence, adjacent sequence elements may also be important for assembly of CELF proteins on RNA by allowing optimal secondary structure to facilitate the formation of RNA-protein complexes [67],[68].

Structural studies have provided valuable insight into the mechanisms underlying the RNA- binding activity of CELF1. CELF proteins all contain two N-terminal and one C-terminal RNA recognition motifs (RRMs), separated by a 160-230 residue divergent domain [69],[70]. The highly conserved RRMs bind to RNA in a sequence-specific manner [69],[71]. Nuclear Magnetic Resonance spectroscopic (NMR)-based solution studies demonstrated that both RRM1 and RRM2 each contribute to binding to a 12-nt target RNA containing two UUGUU motifs. The tandem RRM1/2 domains together show increased affinity compared to the binding by each domain separately to an RNA sequence with two sequential UUGU(U) motifs, thus indicating binding cooperativity between the two RRMs [34],[72]. Crystallographic studies showed that both RRM2 and RRM1 bind to GRE-RNA, and RRM1 is important for crystal-packing interactions [73].

In addition to RRM1 and RRM2, RRM3 also has RNA-binding activity. According to NMR analysis, RRM3 specifically recognizes the UGU trinucleotide segment of bound (UG)3 RNA through extensive stacking and hydrogen-bonding interactions within the pocket formed by the beta-sheet and the conserved N-terminal extension [71]. Experiments investigating CELF1 function through a yeast three hybrid system suggested that deletion/mutation of RRM1 or RRM2 does not abrogate binding to GU-rich RNA, suggesting that RRM3 may recognize GU-repeats more avidly than RRM1 or RRM2 [62]. Additionally, it has been reported that RRM3 is able to recognize a poorly defined G/C-rich sequence from the 5’UTR of Cyclin D1 when combined with the divergent domain [65]. The divergent domain also appears to be important for RNA-binding since the presence of divergent domain within recombinant CELF1/CELF4 chimeric proteins increased RNA-binding affinity, perhaps by conveying important conformational changes necessary for RNA-binding [32],[62],[70]. The divergent domain may also facilitate CELF:CELF homotypic interactions [64] which may influence its activity. For example, CELF:CELF interactions appear to activate RNA deadenylation in Xenopus extracts [66].

3.1. Regulation of CELF1 function through phosphorylation

CELF1 is a known phosphoprotein with multiple predicted phosphorylation sites, and CELF1 phosphorylation appears to regulate its function as a mediator of alternative splicing, mRNA decay, and translational regulation [74],[75],[76],[77]. One of the pathologic events which occurs in the disease Myotonic Dystrophy type 1 (DM1) is an increase in the protein abundance of CELF1 and an associated increase in CELF1 mediated alternative splicing activity. This increase in CELF1 protein abundance is a result of increased CELF1 protein stability secondary to hyperphosphorylation [75]. In DM1, the (CUG)n expansion of the DMPK 3’UTR leads to protein kinase C (PKC) activation through an unknown mechanism. PKC, in turn, hyperphosphorylates CELF1, resulting in increased protein stability and abundance as well as increased splicing activity [78]. Additionally, in transgenic mouse models of DM1, mice treated with specific inhibitors of the PKC pathway showed amelioration of cardiac abnormalities associated with the disease phenotype [79]. Phosphorylation of CELF1 also influences its ability to regulate muscle development (reviewed in [80]). CELF1 phosphorylation by Akt kinase at Ser 28 in normal muscle myoblasts influences its ability to affect the translation of its target transcripts during differentiation [65]. Phosphorylation of CELF1 also directly influences its RNA-binding activity. For example, cyclin D3-Cdk4/6 phosphorylates CELF1 at Ser 302, altering the binding specificity of CELF1 to RNA and translation initiation proteins, such as eIF2α [81]. During the process of T cell activation, phosphorylation of CELF1 alters binding by CELF1 to target transcripts. Shortly following T cell activation, CELF1 becomes phosphorylated, dramatically decreasing its affinity for mRNA and leading to stabilization of CELF1 target transcripts [77]. Overall, these studies show that phosphorylation regulates the many functions of CELF1 in posttranscriptional gene regulation.

3.2. Identification of CELF1 target transcripts

Insight into the biological significance of CELF1 function as a coordinate regulator of post-transcriptional network was revealed through the experimental determination of CELF1 target transcripts. A technique involving RNA-immunoprecipitation followed by microarray analysis of associate transcripts (RIP-Chip) has allowed for the unbiased, genome-wide experimental identification of RNA-binding protein target transcripts. This technique involves immunoprecipitating an RNA-binding protein of interest from cell lysates under conditions that preserve RNA:Protein interactions. The co-purified RNA found associated with the immunoprecipitated RNA-binding protein is then isolated and interrogated using high throughput methods such as microarrays. Using this methodology, CELF1 targets have been identified in HeLa cells, resting and activated human T cells, and mouse myoblasts [55],[77],[82]. CELF1 targets, identified in cytoplasmic extracts from HeLa cells using an anti-CELF1 antibody, were analyzed to identify the CELF1 target sequence, which is known as the GRE. The sequence profile of CELF1 target transcripts was analyzed for enriched sequences using a Markov Chain Monte Carlo based gibbs sampler algorithm (BioProspector) as well as an overrepresentation algorithm, and the previously described GRE sequence, UGUUUGUUUGU, and a GU-repeat sequence, UGUGUGUGUGU, were found to be highly overrepresented in the 3’UTRs of the CELF1 target transcripts [83]. Both sequences were validated as CELF1-binding targets and were shown to function as mRNA decay elements by accelerating the decay of reporter transcripts. While GU-repeat sequences had previously been identified as a CELF1 recognition motif through in vitro SELEX protocol [60]. These and other experiments identified putativebinding targets of CELF1 in cells [32],[62]. Because (as described later in this chapter) the UGUUUGUUUGU sequence and the GU-repeat sequence both bound to CELF1 and functioned as decay elements, the GRE was redefined to contain both of these sequences [83],[84]. The RIP-Chip approach was also used to immunoprecipitate endogenous RNA binding complexes from mouse myoblasts, using an anti-CELF1 antibody and similar G and U rich target sequences were identified [55]. In Xenopus extracts, target transcripts identified by RIP-Chip using an antibody against the CELF1 sequence homolog, EDEN-BP, were enriched in GU-rich sequences very similar to GREs [85]. These GU-rich containing target transcripts represented approximately 5% of the tested transcripts on the microarray [85]. In this work, the authors proposed a 15-nucleotide consensus motif (UGU/UG)3 to be the target motif of EDEN-BP [85],[86]. The RIP-Chip approach was also used to investigate the cytoplasmic target transcripts of CELF1 in resting and activated primary human T cells, and target transcripts were highly enriched for the presence of the GRE in their 3’UTRs, but the number of CELF1 target transcripts decreased dramatically following T cell activation [77]. Overall, numerous CELF1 target transcripts have been identified in several different systems indicating the CELF1 functions to regulate an important posttranscriptional network of gene expression.

Another approach to identify targets of RNA-binding proteins utilizes a cross-linking step prior to immunoprecipitation (CLIP) and subsequent high throughput methods to identify protein binding sequences. Using this method, 315 CELF1 RNA targets were identified in whole cell extracts from mouse hindbrain [87]. These RNA-binding targets for CELF1 were enriched in UG repeat sequences, with 64% of target sequences found in introns and 25% found in 3’ UTR sequences [87]. Similar analysis of CELF1 in the C2C12 mouse myoblast cell line [88] extensively characterized RNA-binding sites of CELF1 and found that CELF1 bound predominantly in 3’UTRs and caused mRNA decay. The authors found significant enrichment of CELF1 binding sites in intronic regions flanking exons, supporting a role for CELF1 in alternative splicing [88]. Overall, these studies suggest that GU-rich sequences serve as genuine binding sites for CELF1 in a manner that has been conserved through evolution. In the next sections, we review the data supporting the model that CELF1 recognizes GU-rich sequences and thereby regulates pre-mRNA splicing, translation, and/or mRNA deadenylation/decay depending on the cellular and environmental context.

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4. CELF1 as a regulator of splicing

Pre-mRNA alternative splicing is a common mechanism for generating transcript and protein diversity. An estimated 90% of human genes produce alternatively spliced transcripts [89],[90]. Alignment of the genomic regions adjacent to mammalian intron-exon splice sites, identified TG-rich motifs (TTCTG and TGTT) as conserved cis-elements found at splicing acceptor sites associated with alternative splicing [91],[92].These C/UG-rich sequences serve as binding sites for CELF proteins which activate or repress the splicing of pre-mRNA targets, depending on the context [93]. Recent evidence has re-confirmed the position-dependence of CELF1-binding sites in regulating exon inclusion or skipping (Figure1) [88]. Although alternative splicing regulation was initially considered the primary function of CELF1 proteins in the nucleus, CELF members have also been implicated in nuclear C to U RNA editing in mammalian cells [94],[95].

Figure 1.

Alternative pre-mRNA splicing by CELF1 protein modulates the temporal and spatial diversity of genes during development. CELF1 binds to GU-rich intronic sequences in precursor mRNA and regulates exon inclusion or exon skipping during stage-specific alternative splicing transitions.

CELF1-mediated regulation of alternative splicing is critical for maintenance of normal muscle structure and function [43],[96],[97]. Much of what we know about the role of CELF1 in alternative splicing comes from studies investigating the role of CELF1 in the pathogenesis of the neuromuscular disease myotonic dystrophy type 1 (DM1). In this disease, aberrant gain of CELF1 function is combined with a corresponding loss of function of the splicing factor MBNL1, resulting in the mis-splicing of a number of crucial genes (reviewed in [11]). Minigene reporter systems that contain alternative splice sites proved to be a useful tools for the identification of pre-mRNA targets for CELF1, including genes for cardiac troponin T (TNNT2)[98], insulin receptor (INSR)[99], and chloride channel1 (CLCN1)[100],[101]. Interestingly, these genes were all shown to be mis-regulated in tissues from patients who suffered from DM1. Minigene systems have been particularly useful in demonstrating that individual pre-mRNA splicing events are affected by loss or gain of activities of specific regulatory proteins. Studies performed in cultured cells with transiently transfected minigenes have identified a number of alternative gene regions regulated by CELF1 and other family members[43],[69],[98],[99],[100],[101],[102],[103],[104],[105],[106],[107],[108]. However, as in other chimeric systems, the results of minigene overexpression experiments may not necessarily reflect the full-length pre-mRNA splicing patterns observed in vivo, especially during certain stages of organism development [109]. CELF proteins have been found to regulate the switch from fetal to adult splicing patterns of several skeletal muscle transcripts through the use of transgenic mouse models[101],[100],[110]. In mice, splicing microarray studies found that nearly half of transcripts that undergo fetal-to-adult alternative splicing transitions in heart respond to over-expression of CELF1, suggesting that the level of CELF1 activity directly regulates the alternative splicing pattern of endogenous transcripts [111]. The development of dominant negative (DN) and tissue specific transgenic mice was advantageous for studying CELF-specific alternative splicing in vivo[96],[104],[109],[110]. For example, dominant negative CELF (DNΔCELF) expressed under the control of a cardiac muscle-specific promoter, promoted the development of dilatated cardiomyopathy and cardiac dysfunction over time [96]. In contrast, when DNΔCELF was expressed under the control of a skeletal muscle-specific promoter, mice exhibit reduction in muscle intersticia and an increase in slow twitch fibers [110]. In near future, we will see more phenotypic studies using a nucleus-restricted form of the dominant negative CELF protein which would specifically block only the CELF1 nuclear function, leaving the cytoplasmic function intact [112], [113], [114].

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5. CELF proteins as regulators of deadenylation, translation, and mRNA decay

5.1. Deadenylation

CELF1 plays important roles in mRNA stability and translation in diverse species. In eukaryotic organisms, the length of a transcript’s polyA tail influences the translational state of a transcript, and deadenylation is regulated by GU-rich sequences and CELF1 proteins across evolution. Regulation of translation through deadenylation in Xenopus embryos is the best characterized model of posttranscriptional gene regulation by CELF proteins. In this model, the shortening or lengthening of the polyA tail causes activation or repression of translation of a number of transcripts [115]. However, as we describe below, deadenylation can also be the first step leading to mRNA degradation in mammalian cells, and the deadenylation machinery seems to be conserved although the consequences of deadenylation (translation or degradation) is regulated differently in diverse species.

In Xenopus, maternal transcripts are stored in the cytoplasm of oocytes in a translationally silent form (reviewed in [116]). After fertilization of Xenopus oocytes, the CELF1 homologue EDEN-BP binds to the EDEN element which activates transcript deadenylation and leads to the translation of EDEN-containing mRNA transcripts, including transcripts that encode important cell cycle regulators [31],[33],[50],[117],[118],[119]. Furthermore, human CELF1, which has 88% identity with EDEN-BP, was able to functionally substitute for EDEN-BP to mediate transcript deadenylation in Xenopus extracts [51], suggesting that the deadenylation function of GU-rich sequences and CELF proteins were conserved in diverse species.

Removal of the polyA tail is the rate-limiting step in the degradation of the majority of mammalian mRNAs [120],[121].In human cell lines, CELF1 has been shown to associate with the deadenylase enzyme polyA ribonuclease (PARN) and to stimulate polyA tail shortening in a cell-free assay using S100 extracts from human cells [52]. It is not known if CELF1 activates other deadenylases in mammalian cells or how deadenylated transcripts are subsequently degraded. PARN, EDEN-BP and cytoplasmic polyadenylation element-binding proteins (CPEB) are present in Xenopus oocyte extracts [122], [123]. Theoretically, the balance between the rate of deadenylation versus polyadenylation depends upon the ability of EDEN-BP to recruit PARN and bind to polyA tail with higher affinity than CPEB [122]. Since EDEN-BP in Xenopus is a regulator of deadenylation, it is likely that CELF1 also regulates deadenylation in mammalian cells, leading to transcript degradation through unknown mechanisms.

5.2. Translation

Translation is a critical layer of post-transcriptional control of gene expression that is regulated in response to environmental and developmental changes. CELF proteins have been shown to be involved in the activation of translation of several mRNA species at various stages of development [124]. Additionally, CELF proteins have been shown to function as inhibitors of translation under conditions of stress, where they act as translational silencers in conjunction with other protein binding partners. The involvement of CELF1 in translational regulation is evolutionarily conserved, with several CELF1 homologues having been shown to regulate translation. For example, in the Drosophila oocyte, translational repression is mediated by the protein Bruno (CELF1 orthologue), that binds specifically to bruno response elements (BREs) within the oskar mRNA 3'UTR. Binding by the CELF1 orthologue Bru-3 to GU-rich sequences in 3’UTR of gurken, cyclin A and oskar mRNA leads to their translational repression [44]. The suggested mechanism underlying Bru-3 mediated translational regulation is through the formation of a Bru-3/eIF4E/5’-cap translational silencing complex during specific stages of embryo development [125]. CELF1 has also been shown to play a role in translational regulation in mammalian cells. In human cell lines, tethering of CELF1 to the 3’UTR of mRNA through an interaction with the MS2 coat protein led to decreased steady state levels of reporter transcripts that contained a MS2 RNA-binding site, while reporter protein levels increased [126]. CELF1 increases the translation of p21 (CDKN1A28) protein [127], and Mef2a29[128]during normal muscle cell differentiationvia direct interaction with (GC)n repeats located within the 5’UTR of those mRNAs. The data suggests that CELF1 mediates translational regulation through interaction with a G- and C-rich motif in the 5’UTR, whereas CELF1 mediates its splicing and degradation effects through interaction with a G- and U-rich motif in introns and 3’UTRs, respectively. Our experiments determining CELF1 binding targets through high-throughput means have failed to identify enrichment of GC-rich motifs or 5’UTR binding by CELF1 [82]. It may be that CELF1 mediated translational regulation is rare, and only occurs in the context of very specific mRNA species and cellular contexts. Recently, an additional mechanism for CELF1 mediated translational regulation through interaction with the 3’UTR was discovered. Binding of CELF1 to the 3’UTR of Serine hydroxymethyltransferase (SHMT) RNA [129],[130] and cyclin dependent kinase inhibitor p27 (Kip1) RNA [131], was found to regulate internal ribosome entry site (IRES) mediated translation activation. This implicated CELF1 in participating in an IRES mode of initiation of mRNA translation. In addition, IRES translation is achieved through CELF1/hnRNPH complex formation, which promotes circularization of RNA transcripts by mediating 5’/3’ ends interactions [129]. Whether CELF1 recruits the translation machinery to the 5’UTR via additional interaction with eIF2(Eukaryotic Initiation Factor 2) or another initiation factor remains to be determined (Figure 2).

Figure 2.

Simplified representation of the role of CELF1 in mRNA translation. Several mechanisms have been proposed for CELF1 mediated regulation of translation. Many of these mechanisms involve CELF1 interacting with mRNA through sequences in the 5’UTR and subsequent translation via an internal ribosomal entry site. If (hyper)/phosphorylated, CELF1 interacts with eIF2 and other translation initiation factors, this serves to promote the production of truncated protein products.

One well studied instance of CELF1 mediated translational control involves the translation of alternative isoforms of the transcription factor CCAAT/enhancer-binding protein (CEBPbeta) [132],[133],[134]. In a rat model, CELF1 phosphorylation was activated by partial hepatectomy, which promoted the formation of a complex between CELF1 and eIF2a. This subsequently led to selective translation of the liver enriched inhibitory protein (LIP) isoform of CCAAT/enhancer-binding protein [76]. It was later shown that in liver, CELF1 undergoes hyper-phosphorylation through a GSK3beta-cyclin D3-cdk4 kinase pathway, and the activity of this pathway seemed to increase with age [135]. Similar to the partial hepatectomy model, the cdk4-mediated hyper-phosphorylation of CELF1 was involved in the age-associated induction of the CELF1-eIF2 complex [136]. In the rat aging model, the CELF1-eIF2 complex binds to the 5’UTR of HDAC1 mRNA and increases histone deacetylase 1 protein levels in aging liver [136],[137]. It was further shown that during rat aging, CELF1 phosphorylation promotes its interaction with a GC-rich sequence in 5’UTR of p21 mRNA causing p21 translational arrest and senescence in fibroblasts [138]. In myocytes, p21 mRNA is stabilized in discrete cytoplasmic structures called stress granules, which serve as reversible storage sites for mRNA under conditions of stress. Interestingly, only during late senescence did p21s localization in stress granules interfere with its translation [138],[139]. One important component of stress granules is the RNA-binding protein T cell internal antigen 1 (TIA1). Consistent with CELF1’s recruitment to stress granules, CELF1 has been shown to function as a translational silencer through interaction with the TIA1 protein [140]. Further support for this model comes from experiments utilizing DM1 cell harboring CUG repeat RNA. The presence of a CUG repeat expansion was found to cause stress and activation of the PKR-phospho-eIF2α–CELF1 pathway leading to stress granule formation and inhibition of mRNA translation [81]. This disruption to physiologic mRNA translation pathways by cellular stress signals might contribute to the progressive muscle loss in DM1 patients. Taken together, this data suggests that CELF proteins may function as activators or repressors of translation, depending on the context.

5.3. mRNA Decay

Bioinformatic analysis of short lived-transcripts in primary human T cells led to the identification of the conserved, GU-rich element (GRE) enriched in transcript’s 3’UTRs. CELF1 was subsequently identified as a protein that specifically bound to the GRE in vitro and then to regulate the decay of exogenously expressed GRE-containing transcripts within cells [30],[141]. Further verification of the role of CELF1 in GRE-mediated mRNA decay came from the observation that in HeLa cells, siRNA-mediated knockdown of CELF1 led to stabilization of GRE-containing beta-globin reporter transcripts, as well as endogenous GRE-containing transcripts [30],[83],[142]. These results implicated CELF1 as a mediator of GRE-dependent mRNA decay. In primary human T cells, GREs and CELF1 appear to be involved in the rapid changes in gene expression patterns observed following T cell receptor-mediated activation. Identification the cytoplasmic binding targets of CELF1 before and after T cell activation led to the discovery that CELF1 dissociated from GRE-containing transcripts following T cell activation in a manner correlated with a transient upregulation of CELF1 target mRNAs [77]. The dissociation of CELF1 from its target transcripts upon T cell activation was the result of an activation-dependent phosphorylation of CELF1 and a resultant decrease in the ability of CELF1 to bind to GRE-containing RNAs [77]. Many of the transiently up-regulated CELF1 target transcripts encoded proteins necessary for the transition from a quiescent state to a state of cellular activation and proliferation. This supported a model whereby CELF1 suppresses a network of transcripts involved in activation and proliferation in resting T cells, and subsequent activation-induced phosphorylation of CELF1 allows for de-repression and accumulation of these transcripts within activated cells.

In mouse myoblasts, cytoplasmic CELF1 bound hundreds of target transcripts that contained GU-rich sequences, including networks of transcripts that regulated cell cycle, intracellular transport and cell survival [55]. Knockdown of CELF1 in this myoblast cell line led to the stabilization of many endogenous GRE-containing targets, as well as luciferase reporter RNAs [88]. Many CELF1 target transcripts were found to be significantly stabilized in CELF1 knockout myoblasts, suggesting that CELF1 mediates the decay of a network of transcripts during myoblast growth and differentiation [55]. In the DM1 disease model, there is aberrant activation of the protein kinase C pathway as a result of the CTG expansion, and this results in CELF1 phosphorylation. Mouse myoblasts (C2C12 cells) made to express CTG expanded RNA were shown to experience stabilization of tumor necrosis factor alpha (TNF-alpha) mRNA [143]. This result suggested that the over-expression of TNF-alpha observed in DM1 could be coming from muscle, and this TNF-alpha overexpression may contribute to the muscle wasting and insulin resistance that are characteristic of this disease [143]. In summary, CELF1 and its GRE-containing target transcripts define posttranscriptional regulatory networks that function to control cellular growth, activation, and differentiation (Figure 3).

Figure 3.

Evolutionary conservation of deadenylation by CELF1 protein and GU-rich sequences. (a). In Xenopus and Drosophila eggs, after fertilization, EDEN-BP (CELF1 homologue) bound to EDEN-containing maternal mRNAs, causing deadenylation and subsequent translational activation.(b). In mammalian cells, CELF1 binds to GREs within the 3' UTR of specific transcripts and promotes their deadenylation (by deadenylases) and subsequent decay by the exosome.

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6. The GRE/CELF1 posttranscriptional network in human diseases

The CELF family is an evolutionarily conserved family of RNA-binding proteins that plays an essential role in several aspects of post-transcriptional gene regulation and participates in the control of important developmental processes. Disruption of CELF1/GRE-mediated mRNA regulation may play a role in the pathophysiology of developmental defects [87],[113],[144], or cancer [145],[146]. In Xenopus, injecting “masking” oligonucleotides into embryos to specifically inhibit the binding of CELF1 to mRNA causes developmental defects, such as the loss of somatic segmentation [147]. Genetic deletion of CELF1 in Caenorhabditis elegans and transgenic mice caused severe developmental abnormalities and death [38],[45]. CELF1 knockout mice were mostly non-viable, but the few surviving pups displayed severe muscular and fertility defects [38]. The finding that CELF1 knockout mice displayed muscle pathophysiology was not surprising since CELF1 was first described as a protein that bound to the abnormally expanded CUG mRNA repeats occurring in patients with the neuromuscular disease: type I myotonic dystrophy [58],[59]. It has since been shown that the molecular pathogenesis of DM1 involves an increase in both nuclear and cytoplasmic CELF1 levels [148],[149] due to hyper-phosphorylation of the protein [74]. Kuyumcu-Martinez and colleagues reported that CELF1 hyper-phosphorylation was triggered by the presence of abnormal CUG repeats in DMPK RNA, which caused cellular stress and a resultant activation of the Protein kinase C stress response pathway. This stress response and CELF1 hyper-phosphorylation was shown to trigger stabilization of the CELF1 protein and thus upregulation in DM1 myoblasts [75]. The importance of CELF1 upregulation is highlighted by the finding that over-expression of CELF1 in mouse heart and skeletal muscle recapitulated many of the aberrant splicing patterns observed in DM1 patient tissues [54],[78],[97],[128],[148],[150]. Interestingly, the repression of CELF1 activity can restore normal alternative splicing events in transgenic mouse model of DM1 [114]. It has become increasingly clear that abnormal splicing underlies the molecular pathogenesis of muscular degenerative disorders, and in addition to occurring in muscle tissue, these splicing changes have been reported in brain tissues [151] which correlated with the presence of neurologic impairment [152] and abnormal Ca(2+) metabolism in DM1 patients [153]. DM1-like alternative splicing dysregulation and altered expression of CELF1 also occurs in mouse models of other muscular dystrophies and muscle injury, most likely due to recapitulation of neonatal splicing patterns in regenerating fibers [113]. CELF1 function is altered in other neuromuscular diseases due to its sequestration to nuclear inclusions in oculopharyngeal muscular dystrophy (OPMD) [154], fragile-X-associated tremor/ataxia syndrome [152], and in spinal bulbar muscular atrophy [155], suggesting a key role for this protein in muscle pathophysiology. It will be interesting to investigate whether altered CELF1 regulation in muscle diseases could also have deleterious effects through altering the stability of GU-rich mRNA targets, given the role of CELF1 in mRNA decay. The discovery of disease-causing splicing patterns in muscle disease has yielded a wealth of information about both physiologic and dysregulated RNA biology and this information is currently being leveraged to develop novel therapies for DM1 and other RNA based neuromuscular disorders [156].

Despite the fact that the field of CELF1 biology is relatively young, there is some data supporting a potential link between dysregulated CELF1 mediated RNA metabolism and a cancerous phenotype. One recent study found CELF1 to be one to the top ten candidates in a transposon-based genetic screen in mice to identify potential drivers of colorectal tumorigenesis [157]. Additionally, CELF1 expression has been shown to be lost through a t(1;11)(q21;q23) translocation in certain forms of pediatric acute leukemia [158]. One way in which disruption of CELF1 may contribute to a malignant phenotype is through disregulation of C/EBPbeta expression. In HER2-overexpressing breast cancer cells CELF1 is activated favoring the production of the C/EBPbeta transcription-inhibitory isoform LIP over that of the active isoform LAP, and this contributed to evasion of TGFbeta and oncogene-induced senescence [146]. Treatment of HER2-transformed metastatic breast cancer cells with the anti-HER2/neu monoclonal antibody trastuzumab reduced CELF1 protein level and it’s activity, suggesting that the targeting of CELF1 may be a viable adjunct therapy in the treatment of breast cancer [159]. Expressions of C/EBPbeta and C/EBPalfaare translationally repressed in BCR/ABL cells (chronic myelogenous leukemia) and it can be re-induced by imatinib via a mechanism that appears to depend on the activity of CELF1 and the integrity of the CUG-rich intercistronic region of C/EBPbeta mRNA[160],[161].

Another potential mechanism of CELF1 mediated tumor promotion comes from our lab’s results of RIP-Chip experiments investigating CELF1’s targets in normal and malignant cells. In primary human T cells, we observed that CELF1 bound to a large number of transcripts involved in cell cycle and apoptosis regulation pathways, and that upon activation and proliferation of these cells, CELF1 bound to a drastically reduced mRNA population [77]. This result suggests that CELF1 inhibition is correlated with a cellular state of proliferation and altered apoptotic response. We also identified hundreds of CELF1 target transcripts in human HeLa cells (carcinoma cell line) and many of these transcripts were different than those in normal T cells suggesting again that altered CELF1’s RNA binding specificity may correlate with malignancy [82].

CELF1-HDAC1-C/EBPbeta pathway is activated in young rat liver cells and in human tumor liver samples suggesting that CELF1-HDAC1-C/EBPbeta complexes are involved in the development of liver tumors [162],[163]. The inhibition of the ubiquitin-dependent proteasome system (UPS) via specific drugs (such as Bortezomib) is one type of approach used to combat cancer [164]. Gareau et. al. showed that CELF1 is required for p21 mRNA stabilization and localization in stress granules induced upon treatment with Bortezomib. The authors postulated that this may allow cancer cells survive stress and escape apoptosis [165]. This mechanism may explain why some tumors are refractory to Bortezomib treatment.

Thus, the dysregulation of CELF1 and GREs appears to contribute to malignant phenotype, perhaps by abrogating its ability to mediate the rapid and timely degradation of GRE-containing growth-regulatory transcripts and promote translation of some cell cycle regulators and oncogenes.

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7. Conclusion

In summary, we have learned a wealth of information about CELF1-mRNA complexes and their importance in development, regeneration, aging and disease. CELF1 binds preferentially to GRE-containing transcripts, and affects expression of transcripts encoding other transcription factors and RNA-binding proteins that regulate cell growth, apoptosis, and development/differentiation (reviewed in [28],[166]). Thus, CELF1 may be functioning as a posttranscriptional “regulator of regulators”, whereby CELF1 influences the expression of a network of target transcripts encoding RNA/DNA binding proteins. This, in turn regulates individual subnetworks of transcripts necessary for development or environmental responses, such as immune activation, requiring transition from a quiescent state to a state of cellular activation and proliferation.

Understanding gene regulatory networks and the integration of transcriptional and posttranscriptional events are the next important tasks in translational medicine. It will require innovations in computational methods, experimental techniques and new animal models. It is also important to further investigate in vivo biochemical interactions between CELF proteins and RNA, to discover unknown components of CELF protein-containing complexes bound to RNA that may be involved in splicing, deadenylation, decay, and/or translation regulation. The lists of conserved RNA-binding proteins and mRNA cis-elements has been expanding over the past decade, but the mechanisms of the precise assembly of RNA-binding complexes in an orchestrated temporal and spatial manner have not been comprehensively described. Furthermore, little work has been done on how the expression and function of CELF1 is regulated, specifically by microRNAs (such as mir-222 [167], mir-503 [168], and miR-23a/b [169]). The more details we learn about intracellular signaling, cross-talk, molecular assembly and localization of RNA-protein complexes, the more unifying principles we may find. Understanding the biochemistry of posttranscriptional regulation will lead to elucidation of posttranscriptional regulatory pathways and networks and lead to a better understanding of normal cellular function and disease states.

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Acknowledgment

This work was supported by NIH grants AIO57484 and AIO72068 to P.R.B. D.B. was supported by MSTP grant T32 GM008244 from the NIH. I.A.V-S. was funded through a fellowship from the Lymphoma Research Foundation.

References

  1. 1. AnticD.JDKeene1997Embryonic lethal abnormal visual RNA-binding proteins involved in growth, differentiation, and posttranscriptional gene expression. Am J Hum Genet 61273278
  2. 2. Jans DA, Xiao CY, Lam MH2000Nuclear targeting signal recognition: a key control point in nuclear transport? Bioessays 22532544
  3. 3. Jansen RP2001mRNA localization: message on the move. Nat Rev Mol Cell Biol 2247256
  4. 4. Faustino NA, Cooper TA2003Pre-mRNA splicing and human disease. Genes Dev 17419437
  5. 5. MataJ.MargueratS.BahlerJ.2005Post-transcriptional control of gene expression: a genome-wide perspective. Trends Biochem Sci 30506514
  6. 6. Moore MJ2005From birth to death: the complex lives of eukaryotic mRNAs. Science 30915141518
  7. 7. Keene JD2007RNA regulons: coordination of post-transcriptional events. Nat Rev Genet 8533543
  8. 8. Mansfield KD, Keene JD2009The ribonome: a dominant force in co-ordinating gene expression. Biol Cell 101169181
  9. 9. Keene JD2007Biological clocks and the coordination theory of RNA operons and regulons. Cold Spring Harb Symp Quant Biol 72157165
  10. 10. MoroyT.HeydF.2007The impact of alternative splicing in vivo: mouse models show the way. RNA 1311551171
  11. 11. Lee JE, Cooper TA2009Pathogenic mechanisms of myotonic dystrophy. Biochem Soc Trans 3712811286
  12. 12. FabianM. R.SonenbergN.FilipowiczW.2010Regulation of mRNA translation and stability by microRNAs. Annu Rev Biochem 79351379
  13. 13. 1623012316Bitel CL, Perrone-Bizzozero NI, Frederikse PH HuB/C/D, nPTB, REST4, and miR-124 regulators of neuronal cell identity are also utilized in the lens. Mol Vis 16: 2301-2316
  14. 14. KeddeM.AgamiR.2008Interplay between microRNAs and RNA-binding proteins determines developmental processes. Cell Cycle 7899903
  15. 15. MooreJ.LaskoP.20 EOF2009Breaking the A chain: regulating mRNAs in development through CCR4 deadenylase. F1000 Biol Rep 1: 20.
  16. 16. BrewerG.2002Messenger RNA decay during aging and development. Ageing Res Rev 1607625
  17. 17. MignoneF.GissiC.LiuniS.PesoleG.2002Untranslated regions of mRNAs. Genome Biol 3: REVIEWS0004 EOF6 EOF
  18. 18. KrolJ.LoedigeI.FilipowiczW.Thewidespread.regulationof.microR. N. A.biogenesisfunction.decayNat Rev Genet 11597610
  19. 19. MisquittaC. M.ChenT.GroverA. K.2006Control of protein expression through mRNA stability in calcium signalling. Cell Calcium 40329346
  20. 20. Khabar KS2007Rapid transit in the immune cells: the role of mRNA turnover regulation. J Leukoc Biol 8113351344
  21. 21. Khabar KS, Young HA2007Post-transcriptional control of the interferon system. Biochimie 89761769
  22. 22. PascaleA.GovoniS.2012The complex world of post-transcriptional mechanisms: is their deregulation a common link for diseases? Focus on ELAV-like RNA-binding proteins. Cell Mol Life Sci 69501517
  23. 23. Tenenbaum SA, Carson CC, Lager PJ, Keene JD2000Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc Natl Acad Sci U S A 971408514090
  24. 24. RaghavanA.OgilvieR. L.ReillyC.AbelsonM. L.RaghavanS.et al.2002Genome-wide analysis of mRNA decay in resting and activated primary human T lymphocytes. Nucleic Acids Res 3055295538
  25. 25. LamL. T.PickeralO. K.PengA. C.RosenwaldA.HurtE. M.et al.2001Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol. Genome Biol 2: RESEARCH0041.
  26. 26. MAFrevelBakheet. T.SilvaA. M.HissongJ. G.KhabarK. S.et al.200338Mitogen-activated protein kinase-dependent and-independent signaling of mRNA stability of AU-rich element-containing transcripts. Mol Cell Biol 23: 425-436.
  27. 27. YangE.van NimwegenE.ZavolanM.RajewskyN.SchroederM.et al.(200Decayrates.ofhuman. m. R. N.Ascorrelationwith.functionalcharacteristics.sequenceattributes.Genome Res 1318631872
  28. 28. Vlasova-StLouis. I.BohjanenP. R.2011Coordinate regulation of mRNA decay networks by GU-rich elements and CELF1. Curr Opin Genet Dev 21444451
  29. 29. VlasovaI. A.Mc NabbJ.RaghavanA.ReillyC.WilliamsD. A.et al.2005Coordinate stabilization of growth-regulatory transcripts in T cell malignancies. Genomics 86159171
  30. 30. VlasovaI. A.TahoeN. M.FanD.LarssonO.RattenbacherB.et al.2008Conserved GU-rich elements mediate mRNA decay by binding to CUG-binding protein 1. Mol Cell 29263270
  31. 31. PaillardL.OmilliF.LegagneuxV.BassezT.ManieyD.et al.1998EDEN and EDEN-BP, a cis element and an associated factor that mediate sequence-specific mRNA deadenylation in Xenopus embryos. EMBO J 17278287
  32. 32. TakahashiN.SasagawaN.SuzukiK.IshiuraS.2000The CUG-binding protein binds specifically to UG dinucleotide repeats in a yeast three-hybrid system. Biochem Biophys Res Commun 277518523
  33. 33. OsborneH. B.Gautier-CourteilleC.GraindorgeA.BarreauC.AudicY.et al.2005Post-transcriptional regulation in Xenopus embryos: role and targets of EDEN-BP. Biochem Soc Trans 3315411543
  34. 34. TripsianesK.SattlerM.2010Repeat recognition. Structure 1812281229
  35. 35. ChoiD. K.ItoT.TsukaharaF.HiraiM.SakakiY.1999Developmentally-regulated expression of mNapor encoding an apoptosis-induced ELAV-type RNA binding protein. Gene 237135142
  36. 36. GoodP. J.ChenQ.WarnerS. J.HerringD. C.2000A family of human RNA-binding proteins related to the Drosophila Bruno translational regulator. J Biol Chem 2752858328592
  37. 37. LiD.BachinskiL. L.RobertsR.2001Genomic organization and isoform-specific tissue expression of human NAPOR (CUGBP2) as a candidate gene for familial arrhythmogenic right ventricular dysplasia. Genomics 74396401
  38. 38. KressC.Gautier-CourteilleC.OsborneH. B.BabinetC.PaillardL.2007Inactivation of CUG-BP1/CELF1 causes growth, viability, and spermatogenesis defects in mice. Mol Cell Biol 2711461157
  39. 39. ChoiD. K.YooK. W.HongS. K.RheeM.SakakiY.et al.2003Isolation and expression of Napor/CUG-BP2 in embryo development. Biochem Biophys Res Commun 305448454
  40. 40. YangY.MahaffeyC. L.BerubeN.MaddatuT. P.CoxG. A.et al.2007Complex seizure disorder caused by Brunol4 deficiency in mice. PLoS Genet 3: e124 EOF
  41. 41. WuJ.LiC.ZhaoS.MaoB.2010Differential expression of the Brunol/CELF family genes during Xenopus laevis early development. Int J Dev Biol 54209214
  42. 42. Morgan GT2007Localized co-transcriptional recruitment of the multifunctional RNA-binding protein CELF1 by lampbrush chromosome transcription units. Chromosome Res 159851000
  43. 43. Ladd AN, Stenberg MG, Swanson MS, Cooper TA2005Dynamic balance between activation and repression regulates pre-mRNA alternative splicing during heart development. Dev Dyn 233783793
  44. 44. Kim-HaJ.KerrK.MacdonaldP. M.1995Translational regulation of oskar mRNA by bruno, an ovarian RNA-binding protein, is essential. Cell 81403412
  45. 45. CAMilneHodgkin. J.1999ETR-1, a homologue of a protein linked to myotonic dystrophy, is essential for muscle development in Caenorhabditis elegans. Curr Biol 912431246
  46. 46. DelaunayJ.Le MeeG.EzzeddineN.LabesseG.TerzianC.et al.2004The Drosophila Bruno paralogue Bru-3 specifically binds the EDEN translational repression element. Nucleic Acids Res 3230703082
  47. 47. HashimotoY.SuzukiH.KageyamaY.YasudaK.InoueK.2006Bruno-like protein is localized to zebrafish germ plasm during the early cleavage stages. Gene Expr Patterns 6201205
  48. 48. Brimacombe KR, Ladd AN (2007) Cloning and embryonic expression patterns of the chicken CELF familyDev Dyn 23622162224
  49. 49. MooreJ.HanH.LaskoP.2009Bruno negatively regulates germ cell-less expression in a BRE-independent manner. Mech Dev 126503516
  50. 50. PaillardL.OsborneH. B.2003East of EDEN was a poly(A) tail. Biol Cell 95211219
  51. 51. PaillardL.LegagneuxV.BeverleyOsborne. H.2003A functional deadenylation assay identifies human CUG-BP as a deadenylation factor. Biol Cell 95107113
  52. 52. MoraesK. C.WiluszC. J.WiluszJ.2006CUG-BP binds to RNA substrates and recruits PARN deadenylase. Rna 1210841091
  53. 53. MichalowskiS.MillerJ. W.UrbinatiC. R.PaliourasM.MSSwansonet.al1999Visualization of double-stranded RNAs from the myotonic dystrophy protein kinase gene and interactions with CUG-binding protein. Nucleic Acids Res 2735343542
  54. 54. WardA. J.RimerM.KillianJ. M.DowlingJ. J.CooperT. A.2010CUGBP1 overexpression in mouse skeletal muscle reproduces features of myotonic dystrophy type 1. Hum Mol Genet 1936143622
  55. 55. LeeJ. E.LeeJ. Y.WiluszJ.TianB.WiluszC. J.2010Systematic analysis of cis-elements in unstable mRNAs demonstrates that CUGBP1 is a key regulator of mRNA decay in muscle cells. PLoS One 5: e11201 EOF14 EOF
  56. 56. SinghG.CharletB. N.HanJ.CooperT. A.2004ETR-3 and CELF4 protein domains required for RNA binding and splicing activity in vivo. Nucleic Acids Res 3212321241
  57. 57. BarreauC.PaillardL.MereauA.OsborneH. B.2006Mammalian CELF/Bruno-like RNA-binding proteins: molecular characteristics and biological functions. Biochimie 88515525
  58. 58. TimchenkoL. T.TimchenkoN. A.CaskeyC. T.RobertsR.1996Novel proteins with binding specificity for DNA CTG repeats and RNA CUG repeats: implications for myotonic dystrophy. Hum Mol Genet 5115121
  59. 59. Timchenko LT, Miller JW, Timchenko NA, DeVore DR, Datar KV, et al.1996Identification of a (CUG)n triplet repeat RNA-binding protein and its expression in myotonic dystrophy. Nucleic Acids Res 2444074414
  60. 60. MarquisJ.PaillardL.AudicY.CossonB.DanosO.et al.2006CUG-BP1/CELF1 requires UGU-rich sequences for high-affinity binding. Biochem J 400291301
  61. 61. GoraczniakR.GundersonS. I.(200Theregulatory.elementin.the3’-untranslated.regionof.humanpapillomavirus. .inhibitsexpression.bybinding. C. U.G-bindingprotein. .J Biol Chem 28322862296
  62. 62. MoriD.SasagawaN.KinoY.IshiuraS.2008Quantitative analysis of CUG-BP1 binding to RNA repeats. J Biochem 143377383
  63. 63. SuzukiH.JinY.OtaniH.YasudaK.InoueK.2002Regulation of alternative splicing of alpha-actinin transcript by Bruno-like proteins. Genes Cells 7133141
  64. 64. Bonnet-CorvenS.AudicY.OmilliF.OsborneH. B.2002An analysis of the sequence requirements of EDEN-BP for specific RNA binding. Nucleic Acids Res 3046674674
  65. 65. SalisburyE.SakaiK.SchoserB.HuichalafC.Schneider-GoldC.et al.(200Ectopicexpression.ofcyclin. D.correctsdifferentiation.ofD. M.myoblaststhrough.activationof. R. N. A. C. U.G-bindingprotein. C. U. G. B. P.Exp Cell Res 31422662278
  66. 66. CossonB.Gautier-CourteilleC.ManieyD.Ait-AhmedO.LesimpleM.et al.2006Oligomerization of EDEN-BP is required for specific mRNA deadenylation and binding. Biol Cell 98653665
  67. 67. WuC.AlwineJ. C.2004Secondary structure as a functional feature in the downstream region of mammalian polyadenylation signals. Mol Cell Biol 2427892796
  68. 68. Mooers BH, Logue JS, Berglund JA2005The structural basis of myotonic dystrophy from the crystal structure of CUG repeats. Proc Natl Acad Sci U S A 1021662616631
  69. 69. LaddA. N.CharletN.CooperT. A.2001The CELF family of RNA binding proteins is implicated in cell-specific and developmentally regulated alternative splicing. Mol Cell Biol 2112851296
  70. 70. HanJ.CooperT. A.2005Identification of CELF splicing activation and repression domains in vivo. Nucleic Acids Res 3327692780
  71. 71. TsudaK.KuwasakoK.TakahashiM.SomeyaT.InoueM.et al.2009Structural basis for the sequence-specific RNA-recognition mechanism of human CUG-BP1 RRM3. Nucleic Acids Res 3751515166
  72. 72. EdwardsJ.MalaurieE.KondrashovA.LongJ.de MoorC. H.et al.2011Sequence determinants for the tandem recognition of UGU and CUG rich RNA elements by the two N--terminal RRMs of CELF1. Nucleic Acids Res 3986388650
  73. 73. TeplovaM.SongJ.GawH. Y.TeplovA.PatelD. J.Structuralinsights.intoR. N. A.recognitionby.thealternate-splicing.regulatorC. U.G-bindingprotein. .Structure 1813641377
  74. 74. RobertsR.TimchenkoN. A.MillerJ. W.ReddyS.CaskeyC. T.et al.1997Altered phosphorylation and intracellular distribution of a (CUG)n triplet repeat RNA-binding protein in patients with myotonic dystrophy and in myotonin protein kinase knockout mice. Proc Natl Acad Sci U S A 941322113226
  75. 75. Kuyumcu-Martinez NM, Wang GS, Cooper TA2007Increased steady-state levels of CUGBP1 in myotonic dystrophy 1 are due to PKC-mediated hyperphosphorylation. Mol Cell 286878
  76. 76. Timchenko NA, Wang GL, Timchenko LT2005RNA CUG-binding protein 1 increases translation of 20-kDa isoform of CCAAT/enhancer-binding protein beta by interacting with the alpha and beta subunits of eukaryotic initiation translation factor 2. J Biol Chem 2802054920557
  77. 77. BeisangD.RattenbacherB.Vlasova-StLouis. I. A.BohjanenP. R.2012Regulation of CUG-binding protein 1 (CUGBP1) binding to target transcripts upon T cell activation. J Biol Chem 287950960
  78. 78. OrengoJ. P.ChambonP.MetzgerD.MosierD. R.SnipesG. J.et al.2008Expanded CTG repeats within the DMPK 3’ UTR causes severe skeletal muscle wasting in an inducible mouse model for myotonic dystrophy. Proc Natl Acad Sci U S A 10526462651
  79. 79. WangG. S.Kuyumcu-MartinezM. N.SarmaS.MathurN.WehrensX. H.et al.2009PKC inhibition ameliorates the cardiac phenotype in a mouse model of myotonic dystrophy type 1. J Clin Invest 11937973806
  80. 80. SchoserB.TimchenkoL.2010Myotonic dystrophies 1 and 2: complex diseases with complex mechanisms. Curr Genomics 117790
  81. 81. HuichalafC.SakaiK.JinB.JonesK.WangG. L.et al.2010Expansion of CUG RNA repeats causes stress and inhibition of translation in myotonic dystrophy 1 (DM1) cells. FASEB J 2437063719
  82. 82. RattenbacherB.BeisangD.WiesnerD. L.JeschkeJ. C.vonHohenberg. M.et al.2010Analysis of CUGBP1 targets identifies GU-repeat sequences that mediate rapid mRNA decay. Mol Cell Biol 3039703980
  83. 83. RattenbacherB.BeisangD.WiesnerD. L.JeschkeJ. C.vonHohenberg. M.et al.Analysisof. C. U. G. B. P.targetsidentifies. G.U-repeatsequences.thatmediate.rapidm. R. N. A.decayMol Cell Biol 3039703980
  84. 84. ASHaleesHitti. E.Al-SaifM.MahmoudL.Vlasova-StLouis. I. A.et al.2011Global assessment of GU-rich regulatory content and function in the human transcriptome. RNA Biol 8681691
  85. 85. GraindorgeA.Le TonquezeO.ThuretR.PolletN.OsborneH. B.et al.(200Identification-Bof. C. U. G.P.-BE. D. E. N.targetP.Asinm. R. N.Xenopustropicalis.Nucleic Acids Res 3618611870
  86. 86. Le TonquezeO.GschloesslB.Namanda-VanderbekenA.LegagneuxV.PaillardL.et al.Chromosome wide analysis of CUGBP1 binding sites identifies the tetraspanin CD9 mRNA as a target for CUGBP1-mediated down-regulation. Biochem Biophys Res Commun 394884889
  87. 87. DaughtersR. S.TuttleD. L.GaoW.IkedaY.MoseleyM. L.et al.2009RNA gain-of-function in spinocerebellar ataxia type 8. PLoS Genet 5: e1000600.
  88. 88. MasudaA.AndersenH. S.DoktorT. K.OkamotoT.ItoM.et al.2012CUGBP1 and MBNL1 preferentially bind to 3’ UTRs and facilitate mRNA decay. Sci Rep 2: 209 EOF
  89. 89. PanQ.ShaiO.LeeL. J.FreyB. J.BlencoweB. J.2008Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet 4014131415
  90. 90. WangE. T.SandbergR.LuoS.KhrebtukovaI.ZhangL.et al.2008Alternative isoform regulation in human tissue transcriptomes. Nature 456470476
  91. 91. Ladd AN, Cooper TA2002Finding signals that regulate alternative splicing in the post-genomic era. Genome Biol 3: reviews0008 EOF
  92. 92. Voelker RB, Berglund JA2007A comprehensive computational characterization of conserved mammalian intronic sequences reveals conserved motifs associated with constitutive and alternative splicing. Genome Res 1710231033
  93. 93. Dembowski JA, Grabowski PJ2009The CUGBP2 splicing factor regulates an ensemble of branchpoints from perimeter binding sites with implications for autoregulation. PLoS Genet 5: e1000595 EOF
  94. 94. AnantS.HendersonJ. O.MukhopadhyayD.NavaratnamN.KennedyS.et al.2001Novel role for RNA-binding protein CUGBP2 in mammalian RNA editing. CUGBP2 modulates C to U editing of apolipoprotein B mRNA by interacting with apobec-1 and ACF, the apobec-1 complementation factor. J Biol Chem 2764733847351
  95. 95. ChenZ.EggermanT. L.PattersonA. P.2007ApoB mRNA editing is mediated by a coordinated modulation of multiple apoB mRNA editing enzyme components. Am J Physiol Gastrointest Liver Physiol 292: G5365
  96. 96. LaddA. N.TaffetG.HartleyC.KearneyD. L.CooperT. A.2005Cardiac tissue-specific repression of CELF activity disrupts alternative splicing and causes cardiomyopathy. Mol Cell Biol 2562676278
  97. 97. HoT. H.BundmanD.ArmstrongD. L.CooperT. A.2005Transgenic mice expressing CUG-BP1 reproduce splicing mis-regulation observed in myotonic dystrophy. Hum Mol Genet 1415391547
  98. 98. Philips AV, Timchenko LT, Cooper TA1998Disruption of splicing regulated by a CUG-binding protein in myotonic dystrophy. Science 280737741
  99. 99. Savkur RS, Philips AV, Cooper TA2001Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat Genet 294047
  100. 100. CharletB. N.SavkurR. S.SinghG.PhilipsA. V.GriceE. A.et al.2002Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. Mol Cell 104553
  101. 101. MankodiA.TakahashiM. P.JiangH.BeckC. L.BowersW. J.et al.2002Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy. Mol Cell 103544
  102. 102. Ladd AN, Cooper TA2004Multiple domains control the subcellular localization and activity of ETR-3, a regulator of nuclear and cytoplasmic RNA processing events. J Cell Sci 11735193529
  103. 103. GromakN.MatlinA. J.CooperT. A.SmithC. W.2003Antagonistic regulation of alpha-actinin alternative splicing by CELF proteins and polypyrimidine tract binding protein. RNA 9443456
  104. 104. CharletB. N.LoganP.SinghG.CooperT. A.2002Dynamic antagonism between ETR-3 and PTB regulates cell type-specific alternative splicing. Mol Cell 9649658
  105. 105. KinoY.WashizuC.OmaY.OnishiH.NezuY.et al.2009MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1. Nucleic Acids Res 3764776490
  106. 106. DujardinG.BurattiE.Charlet-BerguerandN.Martins deAraujo. M.MbopdaA.et al.2010CELF proteins regulate CFTR pre-mRNA splicing: essential role of the divergent domain of ETR-3. Nucleic Acids Res 3872737285
  107. 107. BarronV. A.ZhuH.HinmanM. N.LaddA. N.LouH.2010The neurofibromatosis type I pre-mRNA is a novel target of CELF protein-mediated splicing regulation. Nucleic Acids Res 38253264
  108. 108. KoebisM.OhsawaN.KinoY.SasagawaN.NishinoI.et al.2011Alternative splicing of myomesin 1 gene is aberrantly regulated in myotonic dystrophy type 1. Genes Cells 16961972
  109. 109. TerenziF.BrimacombeK. R.MSPennLadd. A. N.2009CELF-mediated alternative splicing is required for cardiac function during early, but not later, postnatal life. J Mol Cell Cardiol 46395404
  110. 110. DSBergerMoyer. M.KlimentG. M.van LunterenE.LaddA. N.2011Expression of a dominant negative CELF protein in vivo leads to altered muscle organization, fiber size, and subtype. PLoS One 6: e19274 EOF
  111. 111. KalsotraA.XiaoX.WardA. J.CastleJ. C.JohnsonJ. M.et al.2008A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart. Proc Natl Acad Sci U S A 1052033320338
  112. 112. DasguptaT.LaddA. N.2012The importance of CELF control: molecular and biological roles of the CUG-BP, Elav-like family of RNA-binding proteins. Wiley Interdiscip Rev RNA 3104121
  113. 113. Orengo JP, Ward AJ, Cooper TA2011Alternative splicing dysregulation secondary to skeletal muscle regeneration. Ann Neurol 69681690
  114. 114. Berger DS, Ladd AN2012Repression of nuclear CELF activity can rescue CELF-regulated alternative splicing defects in skeletal muscle models of myotonic dystrophy. PLoS Curr 4: RRN1305 EOF
  115. 115. AudicY.OmilliF.OsborneH. B.1998Embryo deadenylation element-dependent deadenylation is enhanced by a cis element containing AUU repeats. Mol Cell Biol 1868796884
  116. 116. JDRichterLasko. P.2011Translational control in oocyte development. Cold Spring Harb Perspect Biol 3: a002758 EOF
  117. 117. JacksonR. J.StandartN.1990Do the poly(A) tail and 3’ untranslated region control mRNA translation? Cell 621524
  118. 118. PaillardL.LegagneuxV.ManieyD.OsborneH. B.2002c-Jun ARE targets mRNA deadenylation by an EDEN-BP (embryo deadenylation element-binding protein)-dependent pathway. J Biol Chem 27732323235
  119. 119. EzzeddineN.PaillardL.CapriM.ManieyD.BassezT.et al.2002EDEN-dependent translational repression of maternal mRNAs is conserved between Xenopus and Drosophila. Proc Natl Acad Sci U S A 99257262
  120. 120. WiluszC. J.WormingtonM.PeltzS. W.2001The cap-to-tail guide to mRNA turnover. Nat Rev Mol Cell Biol 2237246
  121. 121. Chen CY, Shyu AB2011Mechanisms of deadenylation-dependent decay. Wiley Interdiscip Rev RNA 2167183
  122. 122. Kim JH, Richter JD2006Opposing polymerase-deadenylase activities regulate cytoplasmic polyadenylation. Mol Cell 24173183
  123. 123. NovoaI.GallegoJ.FerreiraP. G.MendezR.2010Mitotic cell-cycle progression is regulated by CPEB1 and CPEB4-dependent translational control. Nat Cell Biol 12447456
  124. 124. Horb LD, Horb ME2010BrunoL1 regulates endoderm proliferation through translational enhancement of cyclin A2 mRNA. Dev Biol 345156169
  125. 125. NakamuraA.SatoK.Hanyu-NakamuraK.2004Drosophila cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNA translation in oogenesis. Dev Cell 66978
  126. 126. BarreauC.WatrinT.BeverleyOsborne. H.PaillardL.2006Protein expression is increased by a class III AU-rich element and tethered CUG-BP1. Biochem Biophys Res Commun 347723730
  127. 127. TimchenkoN. A.IakovaP.CaiZ. J.SmithJ. R.TimchenkoL. T.2001Molecular basis for impaired muscle differentiation in myotonic dystrophy. Mol Cell Biol 2169276938
  128. 128. TimchenkoN. A.PatelR.IakovaP.CaiZ. J.QuanL.et al.2004Overexpression of CUG triplet repeat-binding protein, CUGBP1, in mice inhibits myogenesis. J Biol Chem 2791312913139
  129. 129. Fox JT, Stover PJ2009Mechanism of the internal ribosome entry site-mediated translation of serine hydroxymethyltransferase 1. J Biol Chem 2843108531096
  130. 130. WoellerC. F.FoxJ. T.PerryC.StoverP. J.2007A ferritin-responsive internal ribosome entry site regulates folate metabolism. J Biol Chem 2822992729935
  131. 131. ZhengY.MiskiminsW. K.2011CUG-binding protein represses translation of 27Kip1mRNA through its internal ribosomal entry site. RNA Biol 8: 365-371.
  132. 132. TimchenkoN. A.WelmA. L.LuX.TimchenkoL. T.1999CUG repeat binding protein (CUGBP1) interacts with the 5’ region of C/EBPbeta mRNA and regulates translation of C/EBPbeta isoforms. Nucleic Acids Res 2745174525
  133. 133. Bae EJ, Kim SG2005Enhanced CCAAT/enhancer-binding protein beta-liver-enriched inhibitory protein production by Oltipraz, which accompanies CUG repeat-binding protein-1 (CUGBP1) RNA-binding protein activation, leads to inhibition of preadipocyte differentiation. Mol Pharmacol 68660669
  134. 134. KaragiannidesI.ThomouT.TchkoniaT.PirtskhalavaT.KypreosK. E.et al.2006Increased CUG triplet repeat-binding protein-1 predisposes to impaired adipogenesis with aging. J Biol Chem 2812302523033
  135. 135. JinJ.WangG. L.TimchenkoL.TimchenkoN. A.2009GSK3beta and aging liver. Aging (Albany NY) 1582585
  136. 136. TimchenkoL. T.SalisburyE.WangG. L.NguyenH.AlbrechtJ. H.et al.2006Age-specific CUGBP1-eIF2 complex increases translation of CCAAT/enhancer-binding protein beta in old liver. J Biol Chem 2813280632819
  137. 137. JonesK.TimchenkoL.TimchenkoN. A.2012The role of CUGBP1 in age-dependent changes of liver functions. Ageing Res Rev.
  138. 138. IakovaP.WangG. L.TimchenkoL.MichalakM.Pereira-SmithO. M.et al.2004Competition of CUGBP1 and calreticulin for the regulation of 21translation determines cell fate. Embo J 23: 406-417.
  139. 139. Lian XJ, Gallouzi IE2009Oxidative Stress Increases the Number of Stress Granules in Senescent Cells and Triggers a Rapid Decrease in 21waf1cip1 Translation. J Biol Chem 284: 8877-8887.
  140. 140. FujimuraK.KanoF.MurataM.2008Dual localization of the RNA binding protein CUGBP-1 to stress granule and perinucleolar compartment. Exp Cell Res 314543553
  141. 141. Vlasova IA, Bohjanen PR2008Posttranscriptional regulation of gene networks by GU-rich elements and CELF proteins. RNA Biol 5201207
  142. 142. Le TonquezeO.GschloesslB.Namanda-VanderbekenA.LegagneuxV.PaillardL.et al.2010Chromosome wide analysis of CUGBP1 binding sites identifies the tetraspanin CD9 mRNA as a target for CUGBP1-mediated down-regulation. Biochem Biophys Res Commun 394884889
  143. 143. ZhangL.LeeJ. E.WiluszJ.WiluszC. J.2008The RNA-binding protein CUGBP1 regulates stability of tumor necrosis factor mRNA in muscle cells: implications for Myotonic Dystrophy. J Biol Chem.
  144. 144. Philips AV, Cooper TA2000RNA processing and human disease. Cell Mol Life Sci 57235249
  145. 145. La Spada AR, Taylor JP2010Repeat expansion disease: progress and puzzles in disease pathogenesis. Nat Rev Genet 11247258
  146. 146. Arnal-EstapeA.TarragonaM.MoralesM.GuiuM.NadalC.et al.2010HER2 silences tumor suppression in breast cancer cells by switching expression of C/EBPss isoforms. Cancer Res 7099279936
  147. 147. CiboisM.Gautier-CourteilleC.ValleeA.PaillardL.2010A strategy to analyze the phenotypic consequences of inhibiting the association of an RNA-binding protein with a specific RNA. RNA 161015
  148. 148. WangG. S.KearneyD. L.De BiasiM.TaffetG.CooperT. A.2007Elevation of RNA-binding protein CUGBP1 is an early event in an inducible heart-specific mouse model of myotonic dystrophy. J Clin Invest 11728022811
  149. 149. DansithongW.WolfC. M.SarkarP.PaulS.ChiangA.et al.2008Cytoplasmic CUG RNA foci are insufficient to elicit key DM1 features. PLoS One 3: e3968 EOF
  150. 150. KoshelevM.SarmaS.PriceR. E.WehrensX. H.CooperT. A.2010Heart-specific overexpression of CUGBP1 reproduces functional and molecular abnormalities of myotonic dystrophy type 1. Hum Mol Genet 1910661075
  151. 151. LeroyO.WangJ.CAMaurageParent. M.CooperT.et al.2006Brain-specific change in alternative splicing of Tau exon 6 in myotonic dystrophy type 1. Biochim Biophys Acta 1762460467
  152. 152. SofolaO. A.JinP.QinY.DuanR.LiuH.et al.2007RNA-binding proteins hnRNP A2/B1 and CUGBP1 suppress fragile X CGG premutation repeat-induced neurodegeneration in a Drosophila model of FXTAS. Neuron 55565571
  153. 153. HinoS.KondoS.SekiyaH.SaitoA.KanemotoS.et al.2007Molecular mechanisms responsible for aberrant splicing of SERCA1 in myotonic dystrophy type 1. Hum Mol Genet 1628342843
  154. 154. Corbeil-GirardL. P.KleinA. F.SassevilleA. M.LavoieH.MJDicaireet.al2005PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions. Neurobiol Dis 18551567
  155. 155. YuZ.WangA. M.RobinsD. M.LiebermanA. P.2009Altered RNA splicing contributes to skeletal muscle pathology in Kennedy disease knock-in mice. Dis Model Mech 2500507
  156. 156. CooperT. A.WanL.DreyfussG.2009RNA and disease. Cell 136777793
  157. 157. StarrT. K.AllaeiR.SilversteinK. A.StaggsR. A.SarverA. L.et al.2009A transposon-based genetic screen in mice identifies genes altered in colorectal cancer. Science 32317471750
  158. 158. ChoiW. T.FolsomM. R.AzimM. F.MeyerC.KowarzE.et al.2007C/EBPbeta suppression by interruption of CUGBP1 resulting from a complex rearrangement of MLL. Cancer Genet Cytogenet 177108114
  159. 159. Arnal-EstapeA.TarragonaM.MoralesM.GuiuM.NadalC.etal. H. E. R.silencestumor.suppressionin.breastcancer.cellsby.switchingexpression.ofC. E. B.Pssisoforms.Cancer Res 7099279936
  160. 160. GuerzoniC.Ferrari-AmorottiG.BardiniM.MarianiS. A.CalabrettaB.2006Effects of C/EBPalpha and C/EBPbeta in BCR/ABL-expressing cells: differences and similarities. Cell Cycle 512541257
  161. 161. GuerzoniC.BardiniM.MarianiS. A.Ferrari-AmorottiG.NevianiP.et al.2006Inducible activation of CEBPB, a gene negatively regulated by BCR/ABL, inhibits proliferation and promotes differentiation of BCR/ABL-expressing cells. Blood 10740804089
  162. 162. WangG. L.SalisburyE.ShiX.TimchenkoL.EEMedranoet.al2008HDAC1 cooperates with C/EBPalpha in the inhibition of liver proliferation in old mice. J Biol Chem.
  163. 163. WangG. L.SalisburyE.ShiX.TimchenkoL.EEMedranoet.al2008HDAC1 promotes liver proliferation in young mice via interactions with C/EBP beta. J Biol Chem.
  164. 164. MJFournierGareau. C.MazrouiR.2010The chemotherapeutic agent bortezomib induces the formation of stress granules. Cancer Cell Int 10: 12 EOF
  165. 165. GareauC.MJFournierFilion. C.CoudertL.MartelD.et al.201121WAF1/CIP1) upregulation through the stress granule-associated protein CUGBP1 confers resistance to bortezomib-mediated apoptosis. PLoS One 6: e20254.
  166. 166. IakovaP.TimchenkoL.TimchenkoN. A.2011Intracellular signaling and hepatocellular carcinoma. Semin Cancer Biol 212834
  167. 167. XiaoL.CuiY. H.RaoJ. N.ZouT.LiuL.et al.2011Regulation of cyclin-dependent kinase 4 translation through CUG-binding protein 1 and microRNA-222 by polyamines. Mol Biol Cell 2230553069
  168. 168. CuiY. H.XiaoL.RaoJ. N.ZouT.LiuL.et al.2012miR-503 represses CUG-binding protein 1 translation by recruiting CUGBP1 mRNA to processing bodies. Mol Biol Cell 23151162
  169. 169. KalsotraA.WangK.LiP. F.CooperT. A.2010MicroRNAs coordinate an alternative splicing network during mouse postnatal heart development. Genes Dev 24653658

Written By

Daniel Beisang, Paul R. Bohjanen and Irina A. Vlasova-St. Louis

Submitted: 12 January 2012 Published: 19 September 2012