NOVA2-mediated RNA regulation is required for axonal pathfinding during development

The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

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NOVA regulates Dcc alternative splicing during neuronal migration and axon guidance in the spinal cord

eLife ◽

10.7554/elife.14264 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 31

Author(s):

Janelle C Leggere ◽

Yuhki Saito ◽

Robert B Darnell ◽

Marc Tessier-Lavigne ◽

Harald J Junge ◽

...

Keyword(s):

Spinal Cord ◽

Alternative Splicing ◽

Axon Guidance ◽

Neural Development ◽

Neuronal Migration ◽

Rna Binding ◽

Rna Binding Proteins ◽

Splice Variants ◽

Commissural Neurons

RNA-binding proteins (RBPs) control multiple aspects of post-transcriptional gene regulation and function during various biological processes in the nervous system. To further reveal the functional significance of RBPs during neural development, we carried out an in vivo RNAi screen in the dorsal spinal cord interneurons, including the commissural neurons. We found that the NOVA family of RBPs play a key role in neuronal migration, axon outgrowth, and axon guidance. Interestingly, Nova mutants display similar defects as the knockout of the Dcc transmembrane receptor. We show here that Nova deficiency disrupts the alternative splicing of Dcc, and that restoring Dcc splicing in Nova knockouts is able to rescue the defects. Together, our results demonstrate that the production of DCC splice variants controlled by NOVA has a crucial function during many stages of commissural neuron development.

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Targeting Poison Exons to Treat Developmental and Epileptic Encephalopathy

Developmental Neuroscience ◽

10.1159/000516143 ◽

2021 ◽

pp. 1-6

Author(s):

Miriam C. Aziz ◽

Patricia N. Schneider ◽

Gemma L. Carvill

Keyword(s):

Alternative Splicing ◽

Rna Binding ◽

De Novo ◽

Rna Binding Proteins ◽

Autism Spectrum ◽

Epileptic Encephalopathy ◽

Nonsense Mediated Decay ◽

Subsequent Degradation ◽

Alternative Splicing Events ◽

Genetic Epilepsies

Developmental and epileptic encephalopathies (DEEs) describe a subset of neurodevelopmental disorders categorized by refractory epilepsy that is often associated with intellectual disability and autism spectrum disorder. The majority of DEEs are now known to have a genetic basis with de novo coding variants accounting for the majority of cases. More recently, a small number of individuals have been identified with intronic <i>SCN1A</i> variants that result in alternative splicing events that lead to ectopic inclusion of poison exons (PEs). PEs are short highly conserved exons that contain a premature truncation codon, and when spliced into the transcript, lead to premature truncation and subsequent degradation by nonsense-mediated decay. The reason for the inclusion/exclusion of these PEs is not entirely clear, but research suggests an autoregulatory role in gene expression and protein abundance. This is seen in proteins such as RNA-binding proteins and serine/arginine-rich proteins. Recent studies have focused on targeting these PEs as a method for therapeutic intervention. Targeting PEs using antisense oligonucleotides (ASOs) has shown to be effective in modulating alternative splicing events by decreasing the amount of transcripts harboring PEs, thus increasing the abundance of full-length transcripts and thereby the amount of protein in haploinsufficient genes implicated in DEE. In the age of personalized medicine, cellular and animal models of the genetic epilepsies have become essential in developing and testing novel precision therapeutics, including PE-targeting ASOs in a subset of DEEs.

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Evolution of the Small Family of Alternative Splicing Modulators Nuclear Speckle RNA-Binding Proteins in Plants

Genes ◽

10.3390/genes11020207 ◽

2020 ◽

Vol 11 (2) ◽

pp. 207 ◽

Cited By ~ 2

Author(s):

Leandro Lucero ◽

Jeremie Bazin ◽

Johan Rodriguez Melo ◽

Fernando Ibañez ◽

Martín D. Crespi ◽

...

Keyword(s):

Alternative Splicing ◽

Medicago Truncatula ◽

Noncoding Rna ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nodule Development ◽

Nuclear Speckle ◽

Symbiotic Nodule

RNA-Binding Protein 1 (RBP1) was first identified as a protein partner of the long noncoding RNA (lncRNA) ENOD40 in Medicago truncatula, involved in symbiotic nodule development. RBP1 is localized in nuclear speckles and can be relocalized to the cytoplasm by the interaction with ENOD40. The two closest homologs to RBP1 in Arabidopsis thaliana were called Nuclear Speckle RNA-binding proteins (NSRs) and characterized as alternative splicing modulators of specific mRNAs. They can recognize in vivo the lncRNA ALTERNATIVE SPLICING COMPETITOR (ASCO) among other lncRNAs, regulating lateral root formation. Here, we performed a phylogenetic analysis of NSR/RBP proteins tracking the roots of the family to the Embryophytes. Strikingly, eudicots faced a reductive trend of NSR/RBP proteins in comparison with other groups of flowering plants. In Medicago truncatula and Lotus japonicus, their expression profile during nodulation and in specific regions of the symbiotic nodule was compared to that of the lncRNA ENOD40, as well as to changes in alternative splicing. This hinted at distinct and specific roles of each member during nodulation, likely modulating the population of alternatively spliced transcripts. Our results establish the basis to guide future exploration of NSR/RBP function in alternative splicing regulation in different developmental contexts along the plant lineage.

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Neuron-enriched RNA-binding Proteins Regulate Pancreatic Beta Cell Function and Survival

Journal of Biological Chemistry ◽

10.1074/jbc.m116.748335 ◽

2017 ◽

Vol 292 (8) ◽

pp. 3466-3480 ◽

Cited By ~ 24

Author(s):

Jonàs Juan-Mateu ◽

Tatiana H. Rech ◽

Olatz Villate ◽

Esther Lizarraga-Mollinedo ◽

Anna Wendt ◽

...

Keyword(s):

Alternative Splicing ◽

Beta Cell ◽

Beta Cells ◽

Insulin Release ◽

Beta Cell Function ◽

Rna Binding ◽

Cell Function ◽

Rna Binding Proteins ◽

Pancreatic Beta Cell ◽

Splicing Regulators

Pancreatic beta cell failure is the central event leading to diabetes. Beta cells share many phenotypic traits with neurons, and proper beta cell function relies on the activation of several neuron-like transcription programs. Regulation of gene expression by alternative splicing plays a pivotal role in brain, where it affects neuronal development, function, and disease. The role of alternative splicing in beta cells remains unclear, but recent data indicate that splicing alterations modulated by both inflammation and susceptibility genes for diabetes contribute to beta cell dysfunction and death. Here we used RNA sequencing to compare the expression of splicing-regulatory RNA-binding proteins in human islets, brain, and other human tissues, and we identified a cluster of splicing regulators that are expressed in both beta cells and brain. Four of them, namely Elavl4, Nova2, Rbox1, and Rbfox2, were selected for subsequent functional studies in insulin-producing rat INS-1E, human EndoC-βH1 cells, and in primary rat beta cells. Silencing of Elavl4 and Nova2 increased beta cell apoptosis, whereas silencing of Rbfox1 and Rbfox2 increased insulin content and secretion. Interestingly, Rbfox1 silencing modulates the splicing of the actin-remodeling protein gelsolin, increasing gelsolin expression and leading to faster glucose-induced actin depolymerization and increased insulin release. Taken together, these findings indicate that beta cells share common splicing regulators and programs with neurons. These splicing regulators play key roles in insulin release and beta cell survival, and their dysfunction may contribute to the loss of functional beta cell mass in diabetes.

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Transformer 2β Regulates the Alternative Splicing of Cell Cycle Regulator Genes to Promote Ovarian Cancer Cell Progression

10.21203/rs.3.rs-853170/v1 ◽

2021 ◽

Author(s):

Peiying Fu ◽

Ting Zhou ◽

Dong Chen ◽

ShiXuan Wang ◽

Ronghua Liu

Keyword(s):

Cell Cycle ◽

Ovarian Cancer ◽

Alternative Splicing ◽

Cancer Progression ◽

Poor Prognosis ◽

Molecular Mechanisms ◽

Rna Binding ◽

Rna Binding Proteins ◽

Expression Level

Abstract Background: Late-stage ovarian cancer (OV) has a poor prognosis and a high metastasis rate, but the underlying molecular mechanism is ambiguous. RNA binding proteins (RBPs) play important roles in posttranscriptional regulation in the contexts of neoplasia and tumor metastasis. Results: In this study, we explored the molecular functions of a canonical RBP, TRA2B, in cancer cells. TRA2B knockdown in HeLa cells and whole-transcriptome sequencing (RNA-seq) experiments revealed that the TRA2B-regulated alternative splicing (AS) profile was tightly associated with the mitotic cell cycle, apoptosis, and several cancer pathways. Moreover, hundreds of genes were regulated by TRA2B at the expression level, and their functions were enriched in cell proliferation, cell adhesion and angiogenesis, which are related to cancer progression. We also observed that AS regulation and expression regulation occurred independently by integrating the alternatively spliced and differentially expressed genes. We then explored and validated the carcinogenic functions of TRA2B by knocking down its expression in OV cells. In vivo, a high expression level of TRA2B was associated with a poor prognosis in OV patients. Conclusions: We demonstrated the important roles of TRA2B in ovarian neoplasia and OV progression and identified the underlying molecular mechanisms, facilitating the targeted treatment of OV in the future.

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Cardiac Tissue-Specific Repression of CELF Activity Disrupts Alternative Splicing and Causes Cardiomyopathy

Molecular and Cellular Biology ◽

10.1128/mcb.25.14.6267-6278.2005 ◽

2005 ◽

Vol 25 (14) ◽

pp. 6267-6278 ◽

Cited By ~ 49

Author(s):

Andrea N. Ladd ◽

George Taffet ◽

Craig Hartley ◽

Debra L. Kearney ◽

Thomas A. Cooper

Keyword(s):

Alternative Splicing ◽

Cardiac Hypertrophy ◽

Cardiac Fibrosis ◽

Rna Binding ◽

Rna Binding Proteins ◽

Premature Death ◽

Dominant Negative ◽

Splicing Regulation ◽

Protein Activity

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.

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Precise temporal regulation of alternative splicing during neural development

10.1101/247601 ◽

2018 ◽

Author(s):

Sebastien M. Weyn-Vanhentenryck ◽

Huijuan Feng ◽

Dmytro Ustianenko ◽

Rachel Duffié ◽

Qinghong Yan ◽

...

Keyword(s):

Alternative Splicing ◽

Neural Development ◽

Rna Binding ◽

Rna Binding Proteins ◽

Regulatory Mechanisms ◽

Combinatorial Regulation ◽

Temporal Regulation ◽

Maturation Stages

AbstractAlternative splicing (AS) is a crucial step of gene expression that must be tightly controlled, but the precise timing of dynamic splicing switches during neural development and the underlying regulatory mechanisms are poorly understood. Here we systematically analyzed the temporal regulation of AS in a large number of transcriptome profiles of developing mouse cortices, in vivo purified neuronal subtypes, and neurons differentiated in vitro. Our analysis revealed early- and late-switch exons in genes with distinct functions, and these switches accurately define neuronal maturation stages. Integrative modeling suggests that these switches are under direct and combinatorial regulation by distinct sets of neuronal RNA-binding proteins including Nova, Rbfox, Mbnl and Ptbp. Surprisingly, various neuronal subtypes in the sensory systems lack Nova and/or Rbfox expression. These neurons retain the “immature” splicing program in early-switch exons, affecting numerous synaptic genes. These results provide new insights into the organization and regulation of the neurodevelopmental transcriptome.

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The Fox-1 Family and SUP-12 Coordinately Regulate Tissue-Specific Alternative Splicing In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.01508-07 ◽

2007 ◽

Vol 27 (24) ◽

pp. 8612-8621 ◽

Cited By ~ 67

Author(s):

Hidehito Kuroyanagi ◽

Genta Ohno ◽

Shohei Mitani ◽

Masatoshi Hagiwara

Keyword(s):

Alternative Splicing ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Stable Complex ◽

Tissue Specific ◽

Specific Alternative

ABSTRACT Many pre-mRNAs are alternatively spliced in a tissue-specific manner in multicellular organisms. The Fox-1 family of RNA-binding proteins regulate alternative splicing by either activating or repressing exon inclusion through specific binding to UGCAUG stretches. However, the precise cellular contexts that determine the action of the Fox-1 family in vivo remain to be elucidated. We have recently demonstrated that ASD-1 and FOX-1, members of the Fox-1 family in Caenorhabditis elegans, regulate tissue-specific alternative splicing of the fibroblast growth factor receptor gene, egl-15, which eventually determines the ligand specificity of the receptor in vivo. Here we report that another RNA-binding protein, SUP-12, coregulates the egl-15 alternative splicing. By screening for mutants defective in the muscle-specific expression of our alternative splicing reporter, we identified the muscle-specific RNA-binding protein SUP-12. We identified juxtaposed conserved stretches as the cis elements responsible for the regulation. The Fox-1 family and the SUP-12 proteins form a stable complex with egl-15 RNA, depending on the cis elements. Furthermore, the asd-1; sup-12 double mutant is defective in sex myoblast migration, phenocopying the isoform-specific egl-15(5A) mutant. These results establish an in vivo model that coordination of the two families of RNA-binding proteins regulates tissue-specific alternative splicing of a specific target gene.

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Splicing-associated chromatin signatures: a combinatorial and position-dependent role for histone marks in splicing definition

Nature Communications ◽

10.1038/s41467-021-20979-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

E. Agirre ◽

A. J. Oldfield ◽

N. Bellora ◽

A. Segelle ◽

R. F. Luco

Keyword(s):

Alternative Splicing ◽

Rna Binding ◽

Embryonic Stem ◽

Chromatin Modifications ◽

Histone Marks ◽

Splicing Regulators ◽

Machine Learning Approach ◽

Alternatively Spliced ◽

Rna Motif ◽

Regulation Changes

AbstractAlternative splicing relies on the combinatorial recruitment of splicing regulators to specific RNA binding sites. Chromatin has been shown to impact this recruitment. However, a limited number of histone marks have been studied at a global level. In this work, a machine learning approach, applied to extensive epigenomics datasets in human H1 embryonic stem cells and IMR90 foetal fibroblasts, has identified eleven chromatin modifications that differentially mark alternatively spliced exons depending on the level of exon inclusion. These marks act in a combinatorial and position-dependent way, creating characteristic splicing-associated chromatin signatures (SACS). In support of a functional role for SACS in coordinating splicing regulation, changes in the alternative splicing of SACS-marked exons between ten different cell lines correlate with changes in SACS enrichment levels and recruitment of the splicing regulators predicted by RNA motif search analysis. We propose the dynamic nature of chromatin modifications as a mechanism to rapidly fine-tune alternative splicing when necessary.

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Compendium of Methods to Uncover RNA-Protein Interactions In Vivo

Methods and Protocols ◽

10.3390/mps4010022 ◽

2021 ◽

Vol 4 (1) ◽

pp. 22

Author(s):

Mrinmoyee Majumder ◽

Viswanathan Palanisamy

Keyword(s):

Gene Expression ◽

Protein Interactions ◽

Rna Binding ◽

Rna Binding Proteins ◽

Expression Patterns ◽

Control Of Gene Expression ◽

Regulatory Pathways ◽

Advantages And Disadvantages ◽

Protein Nucleic Acid

Control of gene expression is critical in shaping the pro-and eukaryotic organisms’ genotype and phenotype. The gene expression regulatory pathways solely rely on protein–protein and protein–nucleic acid interactions, which determine the fate of the nucleic acids. RNA–protein interactions play a significant role in co- and post-transcriptional regulation to control gene expression. RNA-binding proteins (RBPs) are a diverse group of macromolecules that bind to RNA and play an essential role in RNA biology by regulating pre-mRNA processing, maturation, nuclear transport, stability, and translation. Hence, the studies aimed at investigating RNA–protein interactions are essential to advance our knowledge in gene expression patterns associated with health and disease. Here we discuss the long-established and current technologies that are widely used to study RNA–protein interactions in vivo. We also present the advantages and disadvantages of each method discussed in the review.

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