Andrea N. Ladd

The importance of CELF control: molecular and biological roles of the CUG-BP, Elav-like family of RNA-binding proteins.

RNA processing is important for generating protein diversity and modulating levels of protein expression. The CUG‐BP, Elav‐like family (CELF) of RNA‐binding proteins regulate several steps of RNA processing in the nucleus and cytoplasm, including pre‐mRNA alternative splicing, C to U RNA editing, deadenylation, mRNA decay, and translation. In vivo, CELF proteins have been shown to play roles in gametogenesis and early embryonic development, heart and skeletal muscle function, and neurosynaptic transmission. Dysregulation of CELF‐mediated programs has been implicated in the pathogenesis of human diseases affecting the heart, skeletal muscles, and nervous system. WIREs RNA 2012, 3:104–121. doi: 10.1002/wrna.107

Cardiac tissue-specific repression of CELF activity disrupts alternative splicing and causes cardiomyopathy.

ABSTRACT Members of the CELF family of RNA binding proteins have been implicated in alternative splicing regulation in developing heart. Transgenic mice that express a nuclear dominant-negative CELF protein specifically in the heart (MHC-CELFΔ) develop cardiac hypertrophy and dilated cardiomyopathy with defects in alternative splicing beginning as early as 3 weeks after birth. MHC-CELFΔ mice exhibit extensive cardiac fibrosis, severe cardiac dysfunction, and premature death. Interestingly, the penetrance of the phenotype is greater in females than in males despite similar levels of dominant-negative expression, suggesting that there is sex-specific modulation of splicing activity. The cardiac defects in MHC-CELFΔ mice are directly attributable to reduced levels of CELF activity, as crossing these mice with mice overexpressing CUG-BP1, a wild-type CELF protein, rescues defects in alternative splicing, the severity and incidence of cardiac hypertrophy, and survival. We conclude that CELF protein activity is required for normal alternative splicing in the heart in vivo and that normal CELF-mediated alternative splicing regulation is in turn required for normal cardiac function.

Conserved developmental alternative splicing of muscleblind-like (MBNL) transcripts regulates MBNL localization and activity

Muscleblind-like (MBNL) proteins have been shown to regulate pre-mRNA alternative splicing, and MBNL1 has been implicated in regulating fetal-to-adult transitions in alternative splicing in the heart. MBNL1 is highly conserved, exhibiting more than 95% identity at the amino acid level between birds and mammals. To investigate MBNL1 expression during embryonic heart development, we examined MBNL1 transcript and protein expression in the embryonic chicken heart from the formation of the primitive heart tube through cardiac morphogenesis (embryonic days 1.5 through 8). MBNL1 transcript levels remained steady throughout these stages, whereas MBNL1 protein levels increased and exhibited a shift in isoforms. MBNL1 has several alternatively spliced exons. Using RT-PCR, we determined that the inclusion of one of these, exon 5, decreases dramatically during cardiac morphogenesis. This developmental transition is conserved in mice. Functional analyses of MBNL1 isoforms containing or lacking exon 5-encoded sequences revealed that exon 5 is important for the regulation of the subcellular localization, RNA binding affinity, and alternative splicing activity of MBNL1 proteins. A second MBNL protein, MBNL2, is also expressed in the embryonic heart. We found that MBNL2 exon 5, which is paralogous to MBNL1 exon 5, is similarly regulated during embryonic heart development. Analysis of MBNL1 and MBNL2 transcripts in several embryonic tissues in chicken and mouse indicate that exon 5 alternative splicing is highly conserved and tissue-specific. Thus, we propose that conserved developmental stage- and tissue-specific alternative splicing of MBNL transcripts is an important mechanism by which MBNL activity is regulated during embryonic development.

The neurofibromatosis type I pre-mRNA is a novel target of CELF protein-mediated splicing regulation

The CUG-BP and ETR-3 like factors (CELF) are a family of six highly conserved RNA-binding proteins that preferentially bind to UG-rich sequences. One of the key functions of these proteins is to mediate alternative splicing in a number of tissues, including brain, heart and muscle. To fully understand the function of CELF proteins, it is important to identify downstream targets of CELF proteins. In this communication, we report that neurofibromatosis type I (NF1) exon 23a is a novel target of CELF protein-mediated splicing regulation in neuron-like cells. NF1 regulates Ras signaling, and the isoform that excludes exon 23a shows 10 times greater ability to down-regulate Ras signaling than the isoform that includes exon 23a. Five of the six CELF proteins strongly suppress the inclusion of NF1 exon 23a. Over-expression or siRNA knockdown of these proteins in cell transfection experiments altered the levels of NF1 exon 23a inclusion. In vitro binding and splicing analyses demonstrate that CELF proteins block splicing through interfering with binding of U2AF65. These studies, combined with our previous investigations demonstrating a role for Hu proteins and TIA-1/TIAR in controlling NF1 exon 23a inclusion, highlight the complex nature of regulation of this important alternative splicing event.

RNA binding proteins in the regulation of heart development

In vivo, RNA molecules are constantly accompanied by RNA binding proteins (RBPs), which are intimately involved in every step of RNA biology, including transcription, editing, splicing, transport and localization, stability, and translation. RBPs therefore have opportunities to shape gene expression at multiple levels. This capacity is particularly important during development, when dynamic chemical and physical changes give rise to complex organs and tissues. This review discusses RBPs in the context of heart development. Since the targets and functions of most RBPs--in the heart and at large--are not fully understood, this review focuses on the expression and roles of RBPs that have been implicated in specific stages of heart development or developmental pathology. RBPs are involved in nearly every stage of cardiogenesis, including the formation, morphogenesis, and maturation of the heart. A fuller understanding of the roles and substrates of these proteins could ultimately provide attractive targets for the design of therapies for congenital heart defects, cardiovascular disease, or cardiac tissue repair.

Expression of a Dominant Negative CELF Protein In Vivo Leads to Altered Muscle Organization, Fiber Size, and Subtype

Background CUG-BP and ETR-3-like factor (CELF) proteins regulate tissue- and developmental stage-specific alternative splicing in striated muscle. We previously demonstrated that heart muscle-specific expression of a nuclear dominant negative CELF protein in transgenic mice (MHC-CELFΔ) effectively disrupts endogenous CELF activity in the heart in vivo, resulting in impaired cardiac function. In this study, transgenic mice that express the dominant negative protein under a skeletal muscle-specific promoter (Myo-CELFΔ) were generated to investigate the role of CELF-mediated alternative splicing programs in normal skeletal muscle. Methodology/Principal Findings Myo-CELFΔ mice exhibit modest changes in CELF-mediated alternative splicing in skeletal muscle, accompanied by a reduction of endomysial and perimysial spaces, an increase in fiber size variability, and an increase in slow twitch muscle fibers. Weight gain and mean body weight, total number of muscle fibers, and overall muscle strength were not affected. Conclusions/Significance Although these findings demonstrate that CELF activity contributes to the normal alternative splicing of a subset of muscle transcripts in vivo, the mildness of the effects in Myo-CELFΔ muscles compared to those in MHC-CELFΔ hearts suggests CELF activity may be less determinative for alternative splicing in skeletal muscle than in heart muscle. Nonetheless, even these small changes in CELF-mediated splicing regulation were sufficient to alter muscle organization and muscle fiber properties affected in myotonic dystrophy. This lends further evidence to the hypothesis that dysregulation of CELF-mediated alternative splicing programs may be responsible for the disruption of these properties during muscle pathogenesis.

CELF-mediated alternative splicing is required for cardiac function during early, but not later, postnatal life

During the transition from juvenile to adult life, the heart undergoes programmed remodeling at the levels of transcription and alternative splicing. Members of the CUG-BP and ETR-3-like factor (CELF) family have been implicated in driving developmental transitions in alternative splicing of cardiac transcripts during maturation of the heart. Here, we investigated the timing of the requirement for CELF activity in the postnatal heart using a previously described transgenic mouse model (MHC-CELFDelta). In MHC-CELFDelta mice, nuclear CELF activity has been disrupted specifically in the heart by cardiac-specific expression of a dominant negative CELF protein. Longitudinal analyses of two lines of MHC-CELFDelta mice with differing levels of dominant negative protein expression demonstrate that CELF splicing activity is required for healthy cardiac function during juvenile, but not adult, life. Cardiac function, chamber dilation, and heart size all recover with age in the mild line of MHC-CELFDelta mice without a loss of dominant negative protein expression or change in expression of endogenous CELF proteins or known CELF antagonists. This is the first example of a mouse model with genetically induced cardiomyopathy that spontaneously recovers without intervention. Our results suggest that CELF proteins are key players in the integrated gene expression program involved in postnatal cardiac remodeling and maturation.

Diversity and conservation of CELF1 and CELF2 RNA and protein expression patterns during embryonic development

Introduction: CUG‐BP, Elav‐like family member 1 (CELF1) and CELF2 are RNA‐binding proteins that regulate several stages of RNA processing, and are broadly expressed in developing and adult tissues. In this study, we investigated the expression patterns of CELF1 and CELF2 transcripts and proteins in different tissues, stages of development, and organisms. Results: We found that CELF1 and CELF2 protein levels are regulated independently of transcript levels during heart development, and these proteins exhibit nuclear and cytoplasmic isoforms in the embryonic heart. We found that the subcellular distribution of CELF1 differs between heart, liver, nervous system, and eye, and identified tissue‐specific isoforms of both CELF1 and CELF2 in these tissues. CELF1 and CELF2 are largely co‐expressed, but are found in mutually exclusive territories in several organs, including the heart and eye. Finally, we show that the expression patterns observed in embryonic chicken were mostly recapitulated in the developing mouse, suggesting that the roles of these proteins in the tissues and cells of the developing embryo are conserved as well. Conclusions: CELF1 and CELF2 may underlie conserved, developmentally regulated, tissue‐specific processes in vertebrate embryos. Different tissues likely have unique profiles of nuclear and cytoplasmic CELF1‐ and CELF2‐mediated functions. Developmental Dynamics 242:767–777, 2013.

Muscleblind-like 1 is a negative regulator of TGF-β-dependent epithelial–mesenchymal transition of atrioventricular canal endocardial cells

The development of the valves and septa of the heart depends on the formation and remodeling of endocardial cushions. Here, we report that the alternative splicing regulator muscleblind‐like 1 (MBNL1) exhibits a regionally restricted pattern of expression in canal region endocardium and ventricular myocardium during endocardial cushion development in chicken. Knockdown of MBNL1 in atrioventricular explants leads to a transforming growth factor β‐dependent increase in epithelial–mesenchymal transition (EMT) of endocardial cells. This reveals a novel role for MBNL1 during embryonic development, and represents the first evidence that an alternative splicing regulator is a key player in endocardial cushion development. Developmental Dynamics 238:3266–3272, 2009.

Gene Expression Analyses Implicate an Alternative Splicing Program in Regulating Contractile Gene Expression and Serum Response Factor Activity in Mice

Members of the CUG-BP, Elav-like family (CELF) regulate alternative splicing in the heart. In MHC-CELFΔ transgenic mice, CELF splicing activity is inhibited postnatally in heart muscle via expression of a nuclear dominant negative CELF protein under an α-myosin heavy chain promoter. MHC-CELFΔ mice develop dilated cardiomyopathy characterized by alternative splicing defects, enlarged hearts, and severe contractile dysfunction. In this study, gene expression profiles in the hearts of wild type, high- and low-expressing lines of MHC-CELFΔ mice were compared using microarrays. Gene ontology and pathway analyses identified contraction and calcium signaling as the most affected processes. Network analysis revealed that the serum response factor (SRF) network is highly affected. Downstream targets of SRF were up-regulated in MHC-CELFΔ mice compared to the wild type, suggesting an increase in SRF activity. Although SRF levels remained unchanged, known inhibitors of SRF activity were down-regulated. Conversely, we found that these inhibitors are up-regulated and downstream SRF targets are down-regulated in the hearts of MCKCUG-BP1 mice, which mildly over-express CELF1 in heart and skeletal muscle. This suggests that changes in SRF activity are a consequence of changes in CELF-mediated regulation rather than a secondary result of compensatory pathways in heart failure. In MHC-CELFΔ males, where the phenotype is only partially penetrant, both alternative splicing changes and down-regulation of inhibitors of SRF correlate with the development of cardiomyopathy. Together, these results strongly support a role for CELF-mediated alternative splicing in the regulation of contractile gene expression, achieved in part through modulating the activity of SRF, a key cardiac transcription factor.