Elav-like Family Member Research Articles

Celf1 regulation of dmrt2a is required for somite symmetry and left-right patterning during zebrafish development

RNA-binding proteins (RBPs) bind to numerous and diverse mRNAs to control gene expression post-transcriptionally, although the in vivo functions of specific RBP-mRNA interactions remain largely unknown. Here, we show that an RBP named Cugbp, Elav-like family member 1 (Celf1) controls expression of a gene named doublesex and mab-3 related transcription factor 2a (dmrt2a), which is essential for somite symmetry and left-right patterning during zebrafish development. Celf1 promotes dmrt2a mRNA decay by binding to UGU repeats in the 3'UTR of dmrt2a mRNA such that celf1 overexpression reduces the amount of dmrt2a mRNA, leading to asymmetric somitogenesis and laterality defects. Furthermore, blocking the Celf1-dmrt2a mRNA interaction by a target protector morpholino alleviates failures in somite symmetry and left-right patterning that are caused by celf1 overexpression. Our results therefore demonstrate that Celf1-dependent fine-tuning of dmrt2a expression is essential for generating bilateral symmetry of somites and left-right asymmetric patterning during zebrafish development.

Viral delivery of miR-196a ameliorates the SBMA phenotype via the silencing of CELF2

Spinal and bulbar muscular atrophy (SBMA) is an inherited neurodegenerative disorder caused by the expansion of the polyglutamine (polyQ) tract of the androgen receptor (AR-polyQ). Characteristics of SBMA include proximal muscular atrophy, weakness, contraction fasciculation and bulbar involvement. MicroRNAs (miRNAs) are a diverse class of highly conserved small RNA molecules that function as crucial regulators of gene expression in animals and plants. Recent functional studies have shown the potent activity of specific miRNAs as disease modifiers both in vitro and in vivo. Thus, potential therapeutic approaches that target the miRNA processing pathway have recently attracted attention. Here we describe a novel therapeutic approach using the adeno-associated virus (AAV) vector–mediated delivery of a specific miRNA for SBMA. We found that miR-196a enhanced the decay of the AR mRNA by silencing CUGBP, Elav-like family member 2 (CELF2). CELF2 directly acted on AR mRNA and enhanced the stability of AR mRNA. Furthermore, we found that the early intervention of miR-196a delivered by an AAV vector ameliorated the SBMA phenotypes in a mouse model. Our results establish the proof of principle that disease-specific miRNA delivery could be useful in neurodegenerative diseases.

Repression of nuclear CELF activity can rescue CELF-regulated alternative splicing defects in skeletal muscle models of myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is caused by the expansion of CUG repeats in the 3’ UTR of DMPK transcripts. DM1 pathogenesis has been attributed in part to alternative splicing dysregulation via elevation of CUG-BP, Elav-like family member 1 (CELF1). Several therapeutic approaches have been tested in cells and mice, but no previous studies had specifically targeted CELF1. Here, we show that repressing CELF activity rescues CELF-dependent alternative splicing in cell culture and transgenic mouse models of DM1. CELF-independent splicing, however, remained dysregulated. These data highlight both the potential and limitations of targeting CELF1 for the treatment of DM1.

Reduced Expression of RNA Binding Protein CELF2, a Putative Tumor Suppressor Gene in Colon Cancer

Colon cancer is the third leading cause of cancer death in both men and women in the United States. Every year, 160000 cases of colorectal cancer are diagnosed, and 57000 patients die. CUGBP, Elav-like family member 2 (CELF2) is an RNA binding protein that modulates various posttranscriptional events including RNA splicing, shuttling, editing, stability and translation. Previous studies have demonstrated that CELF2 expression is low in colon cancer cells. Furthermore, ectopic overexpression of CELF2 induces cells to undergo death by mitotic catastrophe. Based on the above observations, we hypothesized that CELF2 expression might be reduced during neoplastic transformation of colon cells. Forty human colon cancer tissues along with 10 uninvolved normal colon tissues from cancer patients were utilized for immunohistochemical analysis of CELF2 expression. We have observed that CELF2 levels are reduced in colon tumor tissues when compared to the normal intestinal tissues. The data set suggests that RNA binding protein CELF2 could be a potential tumor suppressor protein. CELF2 was predominantly nuclear in normal cells, while the cancer tissues had diffused cytoplasmic staining. CELF2 expression is consistently reduced during neoplastic transformation suggesting that it might play a crucial role in tumor initiation and progression.

Etiology of a genetically complex seizure disorder in Celf4 mutant mice

Mice deficient for the gene encoding the RNA-binding protein CELF4 (CUGBP, ELAV-like family member 4) have a complex seizure phenotype that includes both convulsive and non-convulsive seizures, depending upon gene dosage and strain background, modeling genetically complex epilepsy. Invertebrate CELF is associated with translational control in fruit fly ovary epithelium and with neurogenesis and neuronal function in the nematode. Mammalian CELF4 is expressed widely during early development, but is restricted to the central nervous system in adults. To better understand the etiology of the seizure disorder of Celf4 deficient mice, we studied seizure incidence with spatial and temporal conditional knockout Celf4 alleles. For convulsive seizure phenotypes, it is sufficient to delete Celf4 in adulthood at the age of 7 weeks. This timing is in contrast to absence-like non-convulsive seizures, which require deletion before the end of the first postnatal week. Interestingly, selective deletion of Celf4 from cerebral cortex and hippocampus excitatory neurons, but not from inhibitory neurons, is sufficient to lower seizure threshold and to promote spontaneous convulsions. Correspondingly, Celf4 deficient mice have altered excitatory, but not inhibitory, neurotransmission as measured by patch-clamp recordings of cortical layer V pyramidal neurons. Finally, immunostaining in conjunction with an inhibitory neuron-specific reporter shows that CELF4 is expressed predominantly in excitatory neurons. Our results suggest that CELF4 plays a specific role in regulating excitatory neurotransmission. We posit that altered excitatory neurotransmission resulting from Celf4 deficiency underlies the complex seizure disorder in Celf4 mutant mice.

Sequence determinants for the tandem recognition of UGU and CUG rich RNA elements by the two N—terminal RRMs of CELF1

CUGBP, Elav-like family member 1 (CELF1) is an RNA binding protein with important roles in the regulation of splicing, mRNA decay and translation. CELF1 contains three RNA recognition motifs (RRMs). We used gel retardation, gel filtration, isothermal titration calorimetry and NMR titration studies to investigate the recognition of RNA by the first two RRMs of CELF1. NMR shows that RRM1 is promiscuous in binding to both UGU and CUG repeat sequences with comparable chemical shift perturbations. In contrast, RRM2 shows greater selectivity for UGUU rather than CUG motifs. A construct (T187) containing both binding domains (RRM1 and RRM2) was systematically studied for interaction with tandem UGU RNA binding sites with different length linker sequences UGU(U)xUGU where x = 1–7. A single U spacer results in interactions only with RRM1, demonstrating both steric constraints in accommodating both RRMs simultaneously at adjacent sites, and also subtle differences in binding affinities between RRMs. However, high affinity co-operative binding (Kd ~ 0.4 µM) is evident for RNA sequences with x = 2–4, but longer spacers (x ≥ 5) lead to a 10-fold reduction in affinity. Our analysis rationalizes the high affinity interaction of T187 with the 11mer GRE consensus regulatory sequence UGUUUGUUUGU and has significant consequences for the prediction of CELF1 binding sites.

Alternative splicing dysregulation secondary to skeletal muscle regeneration

Dysregulation of alternative splicing has become a molecular hallmark of myotonic dystrophy type 1 (DM1), in which neonatal splice variants are expressed in adult skeletal muscle. Splicing dysregulation is induced by RNA containing expanded CUG repeats expressed from the expanded mutant allele by sequestration of muscleblindlike 1 (MBNL1) protein within nuclear RNA foci and increased CUGBP, ELAV-like family member 1 (CELF1) protein levels. Dysregulated splicing has also been identified in other neuromuscular disorders, suggesting either that diseases with different molecular causes share a common pathogenic mechanism or that dysregulated splicing can also be a common secondary consequence of muscle degeneration and regeneration. In this study, we examined regulation of alternative splicing in 4 different mouse models of muscular dystrophy, including DM1, limb-girdle muscular dystrophy, congenital merosin-deficient muscular dystrophy, and Duchenne muscular dystrophy, and 2 myotoxin (cardiotoxin and notexin) muscle injury models. We show that DM1-like alternative splicing dysregulation and altered expression of MBNL1 and CELF1 occur in non-DM1 mouse models of muscular dystrophy and muscle injury, most likely due to recapitulation of neonatal splicing patterns in regenerating fibers. In contrast, CELF1 was elevated in nuclei of mature myofibers of the DM1 model, consistent with a primary effect of pathogenic RNA expression. Splicing dysregulation in DM1 is a primary effect of RNA containing expanded CUG repeats. However, we conclude that splicing changes can also be observed secondary to muscle regeneration, and this possibility must be taken into account when evaluating cause-effect relationships between dysregulated splicing and disease processes.

Perspectives on gene therapy in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disorder caused by a CTG expansion mutation located in the 3' untranslated region of the DMPK (DM1 protein kinase) gene. According to current evidence, mutant DMPK mRNAs containing the trinucleotide expansion are retained in the nucleus, entrapping Muscleblind (MBNL1) protein and several transcription factors in ribonuclear foci and stabilizing CUG binding protein, Elav-like family member 1 (CELF1), which ultimately causes aberrant pre-mRNA splicing and gene expression of particular genes and associated pathogenesis in patients with DM1. At present, treatment for DM1 is limited to symptomatic intervention, and there is no therapeutic approach to prevent or reverse disease progression. This Mini-Review is focused on the experimental advances obtained in cell-based and animal models toward the development of therapeutic treatments against DM1, providing a discussion of their potential application in clinical trials. Because the central core of DM1 pathogenesis is gain-of-function of mutant RNA, most studies target the mutant RNA by use of antisense oligonucleotides or small chemical compounds to eliminate or ameliorate its toxic effects. However, alternative strategies focused on reversing DM1 features without targeting of mutant DMPK RNA have recently emerged.