A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart

Abstract

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 ...

Four parameters increase the sensitivity and specificity of the exon array analysis and disclose 25 novel aberrantly spliced exons in myotonic dystrophy

“…MBNL1 normally translocates from cytoplasm to nucleus in the postnatal period to induce adult-type splicing, and lack of muscleblind due to sequestration to RNA foci in myotonic dystrophy recapitulates fetal splicing patterns. 21,27 Downregulation of MBNL1 and upregulation of CUGBP1 is likely to occur in rejuvenating muscle fibers, and is likely to result in altered splicing patterns that we observe in disease controls. Not all aberrantly spliced exons in DM1, however, are observed in disease controls.…”

Transcriptionally correlated subcellular dynamics of MBNL1 during lens development and their implication for the molecular pathology of myotonic dystrophy type 1

“…2) MBNL1 has a complex sub-cellular localization in lens epithelial cells Although MBNL1 is recognized as an alternative splicing factor [9,24], additional, extra--nuclear roles for MBNL1 in the regulation of mRNA localization and protein expression have recently been implicated in the pathology of DM1 [8]. In normal HLE cells, endogenous MBNL1 shows a complex subcellular distribution (figure 2A) with staining both in the nucleus and the cytoplasm.…”

Identification of variants in MBNL1 in patients with a myotonic dystrophy-like phenotype

“…[15][16][17][18] For CELF1, multiple functions in RNA metabolism have been reported including regulation of alternative splicing, RNA stability and translational regulation of its RNA targets. [19][20][21] CELF1 interacts with the DMPK transcript, in particular with the (CUG) n repeat. 14 In a mouse model overexpressing CELF1, several typical histological and mis-splicing features of DM were demonstrated.…”

“…MBNL1 is a splicing factor that regulates alternative splicing in development, inducing a switch from a fetal splicing pattern to an adult pattern. 20,23 In DM patients, MBNL1 is bound to the CUG/ CCUG repeat expansion and consequently its activity is impaired, resulting in aberrant alternative splicing of target mRNAs. In a mouse model with deficiency for MBNL1, molecular and phenotypic features of DM were demonstrated, such as myotonia, cataract and splicing defects.…”