From a large-scale screen using splicing microarrays and RT-PCR, we identified 63 alternative splicing (AS) events that are coordinated in 3 distinct temporal patterns during mouse heart development. More than half of these splicing transitions are evolutionarily conserved between mouse and chicken. Computational analysis of the introns flanking these splicing events identified enriched and conserved motifs including binding sites for CUGBP and ETR-3-like factors (CELF), muscleblind-like (MBNL) and Fox proteins. We show that CELF proteins are down-regulated >10-fold during heart development, and MBNL1 protein is concomitantly up-regulated nearly 4-fold. Using transgenic and knockout mice, we show that reproducing the embryonic expression patterns for CUGBP1 and MBNL1 in adult heart induces the embryonic splicing patterns for more than half of the developmentally regulated AS transitions. These findings indicate that CELF and MBNL proteins are determinative for a large subset of splicing transitions that occur during postnatal heart development.CUGBP and ETR-3-like factors ͉ heart development ͉ muscleblind-like ͉ splicing microarray C oordinated control of alternative splicing (AS) on a genomewide scale has the potential to drive proteome transitions with wide-ranging and critical biological consequences (1, 2). Disruption of splicing and its regulation, therefore, is implicated in disease causation and susceptibility (3). Splicing is regulated by RNAbinding proteins that bind to cis-regulatory elements near the splice sites. Some of the best-characterized splicing regulators include the serine-arginine (SR)-rich family, hnRNP proteins, and the Nova, PTB, FOX, TIA, CUGBP and ETR-3-like factors (CELF), and muscleblind-like (MBNL) families (4, 5). CELF and MBNL proteins were first characterized as factors involved in the pathogenesis of myotonic dystrophy and were subsequently shown to be direct regulators of AS (6-8). Recent advances in microarray and computational technologies have allowed comprehensive analyses of individual exons on a genome-wide scale, providing the ability to identify commonly regulated splicing events (9-12).With some exceptions (13,14), most large-scale analyses of regulated splicing have focused primarily on differences between adult tissues and tissues/cell cultures depleted for a splicing regulator rather than normal physiological transitions within a single tissue (9)(10)(11)15). Developmental transitions provide an excellent opportunity to identify and determine the roles for coordinated splicing regulation associated with normal physiological change. The vertebrate heart is particularly attractive for such analysis because it undergoes extensive remodeling to meet the demands of increased workload in the developing organism (16). In addition, the heart has relatively low cellular complexity and little cell turnover (17) so that developmental splicing transitions reflect changes occurring within individual cells to a greater extent than in many other tissues. The physiological changes ...
SUMMARY The p53-regulated long non-coding RNA lincRNA-p21 has been proposed to act in trans via several mechanisms ranging from repressing genes in the p53 transcriptional network to regulating mRNA translation and protein stability. To further examine lincRNA-p21 function we generated a conditional knockout mouse model. We find that lincRNA-p21 predominantly functions in cis to activate expression of its neighboring gene, p21. Mechanistically, we show that lincRNA-p21 acts in concert with hnRNP-K as a co-activator for p53-dependent p21 transcription. Additional phenotypes of lincRNA-p21 deficiency could be attributed to diminished p21 levels, including deregulated expression and altered chromatin state of some Polycomb target genes, defective G1/S checkpoint, increased proliferation rates, and enhanced reprogramming efficiency. These findings indicate that lincRNA-p21 affects global gene expression and influences the p53 tumor suppressor pathway by acting in cis as a locus-restricted co-activator for p53-mediated p21 expression.
Angelman syndrome (AS) is a single gene disorder characterized by intellectual disability, developmental delay, behavioral uniqueness, speech impairment, seizures, and ataxia1,2. It is caused by maternal deficiency of the imprinted gene UBE3A, encoding an E3 ubiquitin ligase3-5. All patients carry at least one copy of paternal UBE3A, which is intact but silenced by a nuclear-localized long non-coding RNA, UBE3A antisense transcript (UBE3A-ATS)6-8. Murine Ube3a-ATS reduction by either transcription termination or topoisomerase I inhibition increased paternal Ube3a expression9,10. Despite a clear understanding of the disease-causing event in AS and the potential to harness the intact paternal allele to correct disease, no gene-specific treatment exists for patients. Here we developed a potential therapeutic intervention for AS by reducing Ube3a-ATS with antisense oligonucleotides (ASOs). ASO treatment achieved specific reduction of Ube3a-ATS and sustained unsilencing of paternal Ube3a in neurons in vitro and in vivo. Partial restoration of UBE3A protein in an AS mouse model ameliorated some cognitive deficits associated with the disease. Although additional studies of phenotypic correction are needed, for the first time we developed a sequence-specific and clinically feasible method to activate expression of the paternal Ube3a allele.
Ninety-four percent of human genes are discontinuous, such that segments expressed as mRNA are contained within exons and separated by intervening segments, called introns. Following transcription, genes are expressed as precursor mRNAs (pre-mRNAs), which are spliced co-transcriptionally, and the flanking exons are joined together to form a continuous mRNA. One advantage of this architecture is that it allows alternative splicing by differential use of exons to generate multiple mRNAs from individual genes. Regulatory elements located within introns and exons guide the splicing complex, the spliceosome, and auxiliary RNA binding proteins to the correct sites for intron removal and exon joining. Misregulation of splicing and alternative splicing can result from mutations in cis-regulatory elements within the affected gene or from mutations that affect the activities of trans-acting factors that are components of the splicing machinery. Mutations that affect splicing can cause disease directly or contribute to the susceptibility or severity of disease. An understanding of the role of splicing in disease expands potential opportunities for therapeutic intervention by either directly addressing the cause or by providing novel approaches to circumvent disease processes.
RNA binding proteins of the conserved CUGBP1, Elav-like factor (CELF) family contribute to heart and skeletal muscle development and are implicated in myotonic dystrophy (DM). To understand their genome-wide functions, we analyzed the transcriptome dynamics following induction of CELF1 or CELF2 in adult mouse heart and of CELF1 in muscle by RNA-seq, complemented by crosslinking/immunoprecipitation-sequencing (CLIP-seq) analysis of mouse cells and tissues to distinguish direct from indirect regulatory targets. We identified hundreds of mRNAs bound in their 3 ′ UTRs by both CELF1 and the developmentally induced MBNL1 protein, a threefold greater overlap in target messages than expected, including messages involved in development and cell differentiation. The extent of 3 ′ UTR binding by CELF1 and MBNL1 predicted the degree of mRNA repression or stabilization, respectively, following CELF1 induction. However, CELF1's RNA binding specificity in vitro was not detectably altered by coincubation with recombinant MBNL1. These findings support a model in which CELF and MBNL proteins bind independently to mRNAs but functionally compete to specify down-regulation or localization/stabilization, respectively, of hundreds of mRNA targets. Expression of many alternative 3 ′ UTR isoforms was altered following CELF1 induction, with 3 ′ UTR binding associated with down-regulation of isoforms and genes. The splicing of hundreds of alternative exons was oppositely regulated by these proteins, confirming an additional layer of regulatory antagonism previously observed in a handful of cases. The regulatory relationships between CELFs and MBNLs in control of both mRNA abundance and splicing appear to have evolved to enhance developmental transitions in major classes of heart and muscle genes.[Supplemental material is available for this article.] CELF RNA binding proteins (RBPs) play roles in early embryonic development, heart, and skeletal muscle functions. They are also thought to contribute to DM pathogenesis (Timchenko et al. 1996) and have been suggested to contribute to other diseases (Ladd 2013). The six family members present in mammals can be divided into two subfamilies: CELF1-2, which are expressed most highly in heart, skeletal muscle, and brain, and CELF3-6, which exhibit more restricted expression (Dasgupta and Ladd 2012). The CELF proteins contain two N-terminal RNA recognition motifs (RRMs) and one C-terminal RRM, with which they bind GU-rich RNAs, and a linker region termed the "divergent domain" that separates RRM2 and RRM3 and is involved in the regulation of alternative pre-mRNA splicing and mRNA decay (Han and Cooper 2005;.During normal development, CELF1 and CELF2 proteins are highly expressed in early embryonic stages and are then down-regulated more than 10-fold in skeletal muscle (Ladd et al. 2005) and the heart (Kalsotra et al. 2008) during post-natal development, remaining at low levels in adult tissues. This developmental downregulation occurs through multiple mechanisms, including repression by microRNAs (m...
During postnatal development the heart undergoes a rapid and dramatic transition to adult function through transcriptional and post-transcriptional mechanisms, including alternative splicing (AS). Here we perform deep RNA-sequencing on RNA from cardiomyocytes and cardiac fibroblasts to conduct a high-resolution analysis of transcriptome changes during postnatal mouse heart development. We reveal extensive changes in gene expression and AS that occur primarily between postnatal days 1 and 28. Cardiomyocytes and cardiac fibroblasts show reciprocal regulation of gene expression reflecting differences in proliferative capacity, cell adhesion functions, and mitochondrial metabolism. We further demonstrate that AS plays a role in vesicular trafficking and membrane organization, These AS transitions are enriched among targets of two RNA-binding proteins, Celf1 and Mbnl1, which undergo developmentally regulated changes in expression. Vesicular trafficking genes affected by AS during normal development (when Celf1 is down-regulated) show a reversion to neonatal splicing patterns after Celf1 re-expression in adults. Short-term Celf1 induction in adult animals results in disrupted transverse tubule organization and calcium handling. These results identify potential roles for AS in multiple aspects of postnatal heart maturation, including vesicular trafficking and intracellular membrane dynamics.
Myotonic dystrophy (dystrophia myotonica, DM) is a multi-systemic disease caused by expanded CTG or CCTG microsatellite repeats. Characterized by symptoms in muscle, heart and central nervous system, among others, it is one of the most variable diseases known. A major pathogenic event in DM is the sequestration of muscleblind-like proteins by CUG or CCUG repeat-containing RNAs transcribed from expanded repeats, and differences in the extent of MBNL sequestration dependent on repeat length and expression level may account for some portion of the variability. However, many other cellular pathways are reported to be perturbed in DM, and the severity of specific disease symptoms varies among individuals. To help understand this variability and facilitate research into DM, we generated 120 RNASeq transcriptomes from skeletal and heart muscle derived from healthy and DM1 biopsies and autopsies. A limited number of DM2 and Duchenne muscular dystrophy samples were also sequenced. We analyzed splicing and gene expression, identified tissue-specific changes in RNA processing and uncovered transcriptome changes strongly correlating with muscle strength. We created a web resource at http://DMseq.org that hosts raw and processed transcriptome data and provides a lightweight, responsive interface that enables browsing of processed data across the genome.
The neuromuscular disease myotonic dystrophy type I (DM1) affects multiple organ systems with the major symptoms being severe muscle weakness, progressive muscle wasting and myotonia. The causative mutation in DM1 is a CTG repeat expansion in the 3'-untranslated region of the DM protein kinase (DMPK) gene. RNA transcribed from the expanded allele contains the expanded CUG repeats and leads to the nuclear depletion of Muscleblind-like 1 (MBNL1) and to the increased steady-state levels of CUG-binding protein 1 (CUGBP1). The pathogenic effects of MBNL1 depletion have previously been tested by the generation of MBNL1 knockout mice, but the consequence of CUGBP1 overexpression in adult muscle is not known. In a DM1 mouse model expressing RNA containing 960 CUG repeats in skeletal muscle, CUGBP1 up-regulation is temporally correlated with severe muscle wasting. In this study, we generated transgenic mice with doxycycline-inducible and skeletal muscle-specific expression of CUGBP1. Adult mouse skeletal muscle overexpressing CUGBP1 reproduces molecular and physiological defects of DM1 tissue. The results from this study strongly suggest that CUGBP1 has a major role in DM1 skeletal muscle pathogenesis.
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