Alternative Splicing Transitions Research Articles

Alternative splicing regulation of membrane trafficking genes during myogenesis.

Alternative splicing transitions occur during organ development, and, in numerous diseases, splicing programs revert to fetal isoform expression. We previously found that extensive splicing changes occur during postnatal mouse heart development in genes encoding proteins involved in vesicle-mediated trafficking. However, the regulatory mechanisms of this splicing-trafficking network are unknown. Here, we found that membrane trafficking genes are alternatively spliced in a tissue-specific manner, with striated muscles exhibiting the highest levels of alternative exon inclusion. Treatment of differentiated muscle cells with chromatin-modifying drugs altered exon inclusion in muscle cells. Examination of several RNA-binding proteins revealed that the poly-pyrimidine tract binding protein 1 (PTBP1) and quaking regulate splicing of trafficking genes during myogenesis, and that removal of PTBP1 motifs prevented PTBP1 from binding its RNA target. These findings enhance our understanding of developmental splicing regulation of membrane trafficking proteins which might have implications for muscle disease pathogenesis.

Alternative splicing transitions associate with emerging atrophy phenotype during denervation-induced skeletal muscle atrophy.

Alternative splicing (AS) presents a key posttranscriptional regulatory mechanism associated with numerous physiological processes. However, little is known about its role in skeletal muscle atrophy. In this study, we used a rat model of denervated skeletal muscle atrophy and performed RNA-sequencing to analyze transcriptome profiling of tibialis anterior muscle at multiple time points following denervation. We found that AS is a novel mechanism involving muscle atrophy, which is independent changes at the transcript level. Bioinformatics analysis further revealed that AS transitions are associated with the appearance of the atrophic phenotype. Moreover, we found that the inclusion of multiple highly conserved exons of Obscn markedly increased at 3 days after denervation. In addition, we confirmed that this newly transcript inhibited C2C12 cell proliferation and exacerbated myotube atrophy. Finally, our study revealed that a large number of RNA-binding proteins were upregulated when the atrophy phenotype appeared. Our data emphasize the importance of AS in this process.

TAMI-49. THE ALTERNATIVE SPLICING FACTOR MBNL1 INHIBITS GLIOBLASTOMA TUMOR INITIATION AND PROGRESSION BY REDUCING HYPOXIA-INDUCED STEMNESS

Abstract Muscleblind-like-proteins (MBNL) belong to a family of tissue-specific RNA metabolism-regulators that control pre-messenger RNA-splicing (AS). Inactivation of MBNL causes an adult-to-fetal AS transition, resulting in the development of myotonic dystrophy. We have previously shown that the aggressive brain cancer, glioblastoma (GBM), maintains stem-like features (GSC) through hypoxia-induced responses. Accordingly, we hypothesized that the hypoxia-induced responses in GBM might also include MBNL-based AS to promote tumor progression. When cultured in hypoxia, GSCs rapidly export MBNL1 out of the nucleus resulting in significant inhibition of MBNL1 activity. Notably, the hypoxia-regulated inhibition of MBNL1 also resulted in evidence of adult-to-fetal alternative splicing transitions. Forced expression of a constitutively active isoform of MBNL1 inhibited GSC self-renewal and tumor initiation in orthotopic transplantation models. Using a tetracycline-inducible system, induced expression of MBNL1 in established orthotopic tumors dramatically inhibited tumor progression resulting in a significant prolongation of survival. This study reveals that MBNL1 plays an essential role in GBM stemness and tumor progression, whereby hypoxic responses within the tumor inhibit MBNL1 activity, promoting stem-like phenotypes and tumor growth. Reversing these effects on MBNL1 may, therefore, yield potent tumor-suppressor activities, uncovering new therapeutic opportunities to counter this devastating disease.

The Alternative Splicing Factor, MBNL1, Inhibits Glioblastoma Tumor Initiation and Progression by Reducing Hypoxia-Induced Stemness.

Muscleblind-like proteins (MBNL) belong to a family of tissue-specific regulators of RNA metabolism that control premessenger RNA splicing. Inactivation of MBNL causes an adult-to-fetal alternative splicing transition, resulting in the development of myotonic dystrophy. We have previously shown that the aggressive brain cancer, glioblastoma (GBM), maintains stem-like features (glioma stem cell, GSC) through hypoxia-induced responses. Accordingly, we hypothesize here that hypoxia-induced responses in GBM might also include MBNL-based alternative splicing to promote tumor progression. When cultured in hypoxia condition, GSCs rapidly exported muscleblind-like-1 (MBNL1) out of the nucleus, resulting in significant inhibition of MBNL1 activity. Notably, hypoxia-regulated inhibition of MBNL1 also resulted in evidence of adult-to-fetal alternative splicing transitions. Forced expression of a constitutively active isoform of MBNL1 inhibited GSC self-renewal and tumor initiation in orthotopic transplantation models. Induced expression of MBNL1 in established orthotopic tumors dramatically inhibited tumor progression, resulting in significantly prolonged survival. This study reveals that MBNL1 plays an essential role in GBM stemness and tumor progression, where hypoxic responses within the tumor inhibit MBNL1 activity, promoting stem-like phenotypes and tumor growth. Reversing these effects on MBNL1 may therefore, yield potent tumor suppressor activities, uncovering new therapeutic opportunities to counter this disease. SIGNIFICANCE: This study describes an unexpected mechanism by which RNA-binding protein, MBNL1, activity is inhibited in hypoxia by a simple isoform switch to regulate glioma stem cell self-renewal, tumorigenicity, and progression.

Extensive alternative splicing transitions during postnatal skeletal muscle development are required for calcium handling functions.

Postnatal development of skeletal muscle is a highly dynamic period of tissue remodeling. Here, we used RNA-seq to identify transcriptome changes from late embryonic to adult mouse muscle and demonstrate that alternative splicing developmental transitions impact muscle physiology. The first 2 weeks after birth are particularly dynamic for differential gene expression and alternative splicing transitions, and calcium-handling functions are significantly enriched among genes that undergo alternative splicing. We focused on the postnatal splicing transitions of the three calcineurin A genes, calcium-dependent phosphatases that regulate multiple aspects of muscle biology. Redirected splicing of calcineurin A to the fetal isoforms in adult muscle and in differentiated C2C12 slows the timing of muscle relaxation, promotes nuclear localization of calcineurin target Nfatc3, and/or affects expression of Nfatc transcription targets. The results demonstrate a previously unknown specificity of calcineurin isoforms as well as the broader impact of alternative splicing during muscle postnatal development.

Abstract 170: Protein Kinase C regulates alternative splicing transitions during heart development

Introduction: Highly conserved genetic and molecular pathways coordinate gene expression during heart development. Alternative splicing (AS) regulates gene expression by generating distinct spliced isoforms of proteins such that embryonic variants are expressed only in fetus but not in adult. Importantly, mutations that affect splicing of embryonic genes such as Nkx2.5 and GATA4 can lead to congenital heart defect. Although AS is critical for proper gene expression in developing heart, the signals that regulate AS are not well known. We have previously shown that Protein Kinase C α/β (PKCα/β) phosphorylates splicing regulator CELF1 that controls AS in heart, suggesting that PKC signaling could be responsible for AS regulation during heart development. Hypothesis: PKCα/β contributes to embryonic gene expression by regulating AS. Methods: We used H9c2 rat embryonic cell differentiation into cardiomyocyte-like cells and rat heart tissues at different stages of development to determine whether PKC is responsible for AS transitions. The localization and levels of proteins were assessed by western blot and immunohistochemistry. Quantitative RT-PCR was performed to determine the splicing pattern of nine developmentally regulated genes using specific primers. Results: The results indicate that AS of developmentally important genes transitioned during rat heart development and H9c2 differentiation. PKCα/β regulated embryonic to neonatal AS transitions by phosphorylating splicing regulators CELF1 and Rbfox2. Blocking PKCα/β activity by bisindolylmaleimide IX reduced both the phosphorylation and protein levels of CELF1 and Rbfox2 with concurrent changes in AS transitions. Depletion of PKCα was sufficient to alter a subset of AS events by down-regulating CELF1 and Rbfox2 proteins. Finally, our data suggest that nuclear localization of PKCα/β at embryonic stages could be the mechanism for AS transitions and increased steady state levels of splicing regulators. Conclusions: Our results show that PKCα/β regulate splicing regulators and AS during heart development. Changes in PKCα/β subcellular localization is one of the determinants for embryonic to neonatal AS transitions in developing rat heart and during cardiomyocyte differentiation.

Functional consequences of developmentally regulated alternative splicing

Genome-wide analyses of metazoan transcriptomes have revealed an unexpected level of mRNA diversity that is generated by alternative splicing. Recently, regulatory networks have been identified through which splicing promotes dynamic remodelling of the transcriptome to promote physiological changes, which involve robust and coordinated alternative splicing transitions. The regulation of splicing in yeast, worms, flies and vertebrates affects a variety of biological processes. The functional classes of genes that are regulated by alternative splicing include both those with widespread homeostatic activities and those with cell-type-specific functions. Alternative splicing can drive determinative physiological change or can have a permissive role by providing mRNA variability that is used by other regulatory mechanisms.

Analysis of Exonic Regions Involved in Nuclear Localization, Splicing Activity, and Dimerization of Muscleblind-like-1 Isoforms

Muscleblind-like-1 (MBNL1) is a splicing regulatory factor controlling the fetal-to-adult alternative splicing transitions during vertebrate muscle development. Its capture by nuclear CUG expansions is one major cause for type 1 myotonic dystrophy (DM1). Alternative splicing produces MBNL1 isoforms that differ by the presence or absence of the exonic regions 3, 5, and 7. To understand better their respective roles and the consequences of the deregulation of their expression in DM1, here we studied the respective roles of MBNL1 alternative and constitutive exons. By combining genetics, molecular and cellular approaches, we found that (i) the exon 5 and 6 regions are both needed to control the nuclear localization of MBNL1; (ii) the exon 3 region strongly enhances the affinity of MBNL1 for its pre-mRNA target sites; (iii) the exon 3 and 6 regions are both required for the splicing regulatory activity, and this function is not enhanced by an exclusive nuclear localization of MBNL1; and finally (iv) the exon 7 region enhances MBNL1-MBNL1 dimerization properties. Consequently, the abnormally high inclusion of the exon 5 and 7 regions in DM1 is expected to enhance the potential of MBNL1 of being sequestered with nuclear CUG expansions, which provides new insight into DM1 pathophysiology.

MicroRNAs coordinate an alternative splicing network during mouse postnatal heart development

Alternative splicing transitions have been identified recently as a conserved component of vertebrate heart remodeling during postnatal development. Here we report that the targeted deletion of Dicer, specifically in adult mouse myocardium, reveals the role of microRNAs (miRNAs) in regulating networks of postnatal splicing transitions and in maintaining adult splicing programs. We demonstrate a direct role for miR-23a/b in the dramatic postnatal down-regulation of CUGBP and ETR-3-like factor (CELF) proteins that regulate nearly half of developmentally regulated splicing transitions in the heart. These findings define a hierarchy in which rapid postnatal up-regulation of specific miRNAs controls expression of alternative splicing regulators and the subsequent splicing transitions of their downstream targets.

Heart-specific overexpression of CUGBP1 reproduces functional and molecular abnormalities of myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is caused by a CTG expansion within the 3'-untranslated region of the DMPK gene. The predominant mechanism of pathogenesis is a toxic gain of function of CUG repeat containing RNA transcribed from the expanded allele. The molecular mechanisms by which the RNA containing expanded repeats produce pathogenic effects include: sequestration of muscleblind-like 1 (MBNL1) protein and up-regulation of CUG binding protein 1 (CUGBP1). MBNL1 and CUGBP1 are RNA binding proteins that regulate alternative splicing transitions during development. Altered functions of these proteins in DM1 lead to misregulated splicing of their target genes, resulting in several features of the disease. The role of MBNL1 depletion in DM1 is well established through a mouse knock-out model that reproduces many disease features. Here we directly test the hypothesis that CUGBP1 up-regulation also contributes to manifestations of DM1. Using tetracycline-inducible CUGBP1 and heart-specific reverse tetracycline trans-activator transgenes, we expressed human CUGBP1 in adult mouse heart. Our results demonstrate that up-regulation of CUGBP1 is sufficient to reproduce molecular, histopathological and functional changes observed in a previously described DM1 mouse model that expresses expanded CUG RNA repeats as well as in individuals with DM1. These results strongly support a role for CUGBP1 up-regulation in DM1 pathogenesis.