Splicing regulation through biomolecular condensates and membraneless organelles.

Biomolecular condensates, sometimes also known as membraneless organelles (MLOs), can form through weak multivalent intermolecular interactions of proteins and nucleic acids, a process often associated with liquid-liquid phase separation. Biomolecular condensates are emerging as sites and regulatory platforms of vital cellular functions, including transcription and RNA processing. In the first part of this Review, we comprehensively discuss how alternative splicing regulates the formation and properties of condensates, and conversely the roles of biomolecular condensates in splicing regulation. In the second part, we focus on the spatial connection between splicing regulation and nuclear MLOs such as transcriptional condensates, splicing condensates and nuclear speckles. We then discuss key studies showing how splicing regulation through biomolecular condensates is implicated in human pathologies such as neurodegenerative diseases, different types of cancer, developmental disorders and cardiomyopathies, and conclude with a discussion of outstanding questions pertaining to the roles of condensates and MLOs in splicing regulation and how to experimentally study them.

Alternative splicing as a regulator of development and tissue identity.

Alternative splicing of eukaryotic transcripts is a mechanism that enables cells to generate vast protein diversity from a limited number of genes. The mechanisms and outcomes of alternative splicing of individual transcripts are relatively well understood, and recent efforts have been directed towards studying splicing networks. It has become apparent that coordinated splicing networks regulate tissue and organ development, and that alternative splicing has important physiological functions in different developmental processes in humans.

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.

The synaptosome-associated protein 23 (SNAP23) is necessary for proper myogenesis.

Vesicle-mediated transport is necessary for maintaining cellular homeostasis and proper signaling. The synaptosome-associated protein 23 (SNAP23) is a member of the SNARE protein family and mediates the vesicle docking and membrane fusion steps of secretion during exocytosis. Skeletal muscle has been established as a secretory organ; however, the role of SNAP23 in the context of skeletal muscle development is still unknown. Here, we show that depletion of SNAP23 in C2C12 mouse myoblasts reduces their ability to differentiate into myotubes as a result of premature cell cycle exit and early activation of the myogenic transcriptional program. This effect is rescued when cells are seeded at a high density or when cultured in conditioned medium from wild type cells. Proteomic analysis of collected medium indicates that SNAP23 depletion leads to a misregulation of exocytosis, including decreased secretion of the insulin-like growth factor 1 (IGF1), a critical protein for muscle growth, development, and function. We further demonstrate that treatment of SNAP23-depleted cells with exogenous IGF1 rescues their myogenic capacity. We propose that SNAP23 mediates the secretion of specific proteins, such as IGF1, that are important for achieving proper differentiation of skeletal muscle cells during myogenesis. This work highlights the underappreciated role of skeletal muscle as a secretory organ and contributes to the understanding of factors necessary for myogenesis.

How alternative splicing affects membrane-trafficking dynamics.

The cell biology field has outstanding working knowledge of the fundamentals of membrane-trafficking pathways, which are of critical importance in health and disease. Current challenges include understanding how trafficking pathways are fine-tuned for specialized tissue functions in vivo and during development. In parallel, the ENCODE project and numerous genetic studies have revealed that alternative splicing regulates gene expression in tissues and throughout development at a post-transcriptional level. This Review summarizes recent discoveries demonstrating that alternative splicing affects tissue specialization and membrane-trafficking proteins during development, and examines how this regulation is altered in human disease. We first discuss how alternative splicing of clathrin, SNAREs and BAR-domain proteins influences endocytosis, secretion and membrane dynamics, respectively. We then focus on the role of RNA-binding proteins in the regulation of splicing of membrane-trafficking proteins in health and disease. Overall, our aim is to comprehensively summarize how trafficking is molecularly influenced by alternative splicing and identify future directions centered on its physiological relevance.

Heme Oxygenase 1 Impairs Glucocorticoid Receptor Activity in Prostate Cancer.

Glucocorticoids are used during prostate cancer (PCa) treatment. However, they may also have the potential to drive castration resistant prostate cancer (CRPC) growth via the glucocorticoid receptor (GR). Given the association between inflammation and PCa, and the anti-inflammatory role of heme oxygenase 1 (HO-1), we aimed at identifying the molecular processes governed by the interaction between HO-1 and GR. PCa-derived cell lines were treated with Hemin, Dexamethasone (Dex), or both. We studied GR gene expression by RTqPCR, protein expression by Western Blot, transcriptional activity using reporter assays, and nuclear translocation by confocal microscopy. We also evaluated the expression of HO-1, FKBP51, and FKBP52 by Western Blot. Hemin pre-treatment reduced Dex-induced GR activity in PC3 cells. Protein levels of FKBP51, a cytoplasmic GR-binding immunophilin, were significantly increased in Hemin+Dex treated cells, possibly accounting for lower GR activity. We also evaluated these treatments in vivo using PC3 tumors growing as xenografts. We found non-significant differences in tumor growth among treatments. Immunohistochemistry analyses revealed strong nuclear GR staining in almost all groups. We did not observe HO-1 staining in tumor cells, but high HO-1 reactivity was detected in tumor infiltrating macrophages. Our results suggest an association and crossed modulation between HO-1 and GR pathways.

Antagonistic regulation of mRNA expression and splicing by CELF and MBNL proteins.

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.

The RNA-binding protein Rbfox1 regulates splicing required for skeletal muscle structure and function.

The Rbfox family of RNA-binding proteins is highly conserved with established roles in alternative splicing (AS) regulation. High-throughput studies aimed at understanding transcriptome remodeling have revealed skeletal muscle as displaying one of the largest number of AS events. This finding is consistent with requirements for tissue-specific protein isoforms needed to sustain muscle-specific functions. Rbfox1 is abundant in vertebrate brain, heart and skeletal muscle. Genome-wide genetic approaches have linked the Rbfox1 gene to autism, and a brain-specific knockout mouse revealed a critical role for this splicing regulator in neuronal function. Moreover, a Caenorhabditis elegans Rbfox1 homolog regulates muscle-specific splicing. To determine the role of Rbfox1 in muscle function, we developed a conditional knockout mouse model to specifically delete Rbfox1 in adult tissue. We show that Rbfox1 is required for muscle function but a >70% loss of Rbfox1 in satellite cells does not disrupt muscle regeneration. Deep sequencing identified aberrant splicing of multiple genes including those encoding myofibrillar and cytoskeletal proteins, and proteins that regulate calcium handling. Ultrastructure analysis of Rbfox1(-/-) muscle by electron microscopy revealed abundant tubular aggregates. Immunostaining showed mislocalization of the sarcoplasmic reticulum proteins Serca1 and Ryr1 in a pattern indicative of colocalization with the tubular aggregates. Consistent with mislocalization of Serca1 and Ryr1, calcium handling was drastically altered in Rbfox1(-/-) muscle. Moreover, muscle function was significantly impaired in Rbfox1(-/-) muscle as indicated by decreased force generation. These results demonstrate that Rbfox1 regulates a network of AS events required to maintain multiple aspects of muscle physiology.

RNA-binding proteins in heart development.

RNA-binding proteins (RBPs) are key players of posttranscriptional regulation occurring during normal tissue development. All tissues examined thus far have revealed the importance of RBPs in the regulation of complex networks involved in organ morphogenesis, maturation, and function. They are responsible for controlling tissue-specific gene expression by regulating alternative splicing, mRNA stability, translation, and poly-adenylation. The heart is the first organ form during embryonic development and is also the first to acquire functionality. Numerous remodeling processes take place during late cardiac development since fetal heart first adapts to birth and then undergoes a transition to adult functionality. This physiological remodeling involves transcriptional and posttranscriptional networks that are regulated by RBPs. Disruption of the normal regulatory networks has been shown to cause cardiomyopathy in humans and animal models. Here we review the complexity of late heart development and the current information regarding how RBPs control aspects of postnatal heart development. We also review how activities of RBPs are modulated adding complexity to the regulation of developmental networks.

Differential endocytosis and signaling dynamics of insulin receptor variants IR-A and IR-B.

Insulin signaling comprises a complex cascade of events, playing a key role in the regulation of glucose metabolism and cellular growth. Impaired response to insulin is the hallmark of diabetes, whereas upregulated insulin activity occurs in many cancers. Two splice variants of the insulin receptor (IR) exist in mammals: IR-A, lacking exon 11, and full-length IR-B. Although considerable biochemical data exist on insulin binding and downstream signaling, little is known about the dynamics of the IR itself. We created functional IR transgenes fused with visible fluorescent proteins for use in combination with biotinamido-caproyl insulin and streptavidin quantum dots. Using confocal and structured illumination microscopy, we visualized the endocytosis of both isoforms in living and fixed cells and demonstrated a higher rate of endocytosis of IR-A than IR-B. These differences correlated with higher and sustained activation of IR-A in response to insulin and with distinctive ERK1/2 activation profiles and gene transcription regulation. In addition, cells expressing IR-B showed higher AKT phosphorylation after insulin stimulation than cells expressing IR-A. Taken together, these results suggest that IR signaling is dependent on localization; internalized IRs regulate mitogenic activity, whereas metabolic balance signaling occurs at the cell membrane.

Endocytosis and intracellular dissociation rates of human insulin-insulin receptor complexes by quantum dots in living cells.

Insulin signaling is involved in glucose metabolism, cellular growth, and differentiation. Its function is altered in diabetes and many cancer types. Insulin binding to insulin receptor (IR) triggers diverse signaling pathways. However, signal transduction by IR is not mediated exclusively at the cell surface. Activated ligand-receptor complexes are internalized into endosomes from which the IR recruits adapters acting on substrates that are distinct from those accessible at the membrane. We report the biotinylation of human-recombinant insulin (rhIns) specifically at the position 29 of the B chain. We combined visible fluorescent proteins fused to IR and biotinylated rhIns conjugated with streptavidin-quantum dots to perform extended, quantitative experiments in real time. Modified rhIns bound to the IR and conjugated with the quantum dots was internalized with a rate constant (k) of 0.009 min(-1). Dissociation of insulin-IR complex in endocytosed vesicles occurred with k = 0.006 min(-1).

Insulin receptor membrane retention by a traceable chimeric mutant.

BACKGROUND: The insulin receptor (IR) regulates glucose homeostasis, cell growth and differentiation. It has been hypothesized that the specific signaling characteristics of IR are in part determined by ligand-receptor complexes localization. Downstream signaling could be triggered from the plasma membrane or from endosomes. Regulation of activated receptor's internalization has been proposed as the mechanism responsible for the differential isoform and ligand-specific signaling. RESULTS: We generated a traceable IR chimera that allows the labeling of the receptor at the cell surface. This mutant binds insulin but fails to get activated and internalized. However, the mutant heterodimerizes with wild type IR inhibiting its auto-phosphorylation and blocking its internalization. IR membrane retention attenuates AP-1 transcriptional activation favoring Akt activation. CONCLUSIONS: These results suggest that the mutant acts as a selective dominant negative blocking IR internalization-mediated signaling.

Insulin and insulin like growth factor II endocytosis and signaling via insulin receptor B.

BACKGROUND: Insulin and insulin-like growth factors (IGFs) act on tetrameric tyrosine kinase receptors controlling essential functions including growth, metabolism, reproduction and longevity. The insulin receptor (IR) binds insulin and IGFs with different affinities triggering different cell responses. RESULTS: We showed that IGF-II induces cell proliferation and gene transcription when IR-B is over-expressed. We combined biotinylated ligands with streptavidin conjugated quantum dots and visible fluorescent proteins to visualize the binding of IGF-II and insulin to IR-B and their ensuing internalization. By confocal microscopy and flow cytometry in living cells, we studied the internalization kinetic through the IR-B of both IGF-II, known to elicit proliferative responses, and insulin, a regulator of metabolism. CONCLUSIONS: IGF-II promotes a faster internalization of IR-B than insulin. We propose that IGF-II differentially activates mitogenic responses through endosomes, while insulin-activated IR-B remains at the plasma membrane. This fact could facilitate the interaction with key effector molecules involved in metabolism regulation.

RBFOX2 regulated EYA3 isoforms partner with SIX4 or ZBTB1 to control transcription during myogenesis.

Alternative splicing is a prevalent gene-regulatory mechanism, with over 95% of multi-exon human genes estimated to be alternatively spliced. Here, we describe a tissue-specific, developmentally regulated, highly conserved, and disease-associated alternative splicing event in exon 7 of the eyes absent homolog 3 (Eya3) gene. We discovered that EYA3 expression is vital to the proliferation and differentiation of myoblasts. Genome-wide transcriptomic analysis and mass spectrometry-based proteomic studies identified SIX homeobox 4 (SIX4) and zinc finger and BTB-domain containing 1 (ZBTB1), as major transcription factors that interact with EYA3 to dictate gene expression. EYA3 isoforms differentially regulate transcription, indicating that splicing aids in temporal control of gene expression during muscle cell differentiation. Finally, we identified RNA-binding fox-1 homolog 2 (RBFOX2) as the main regulator of EYA3 splicing. Together, our findings illustrate the interplay between alternative splicing and transcription during myogenesis.

SET domain containing 2 (SETD2) influences metabolism and alternative splicing during myogenesis.

Epigenetic regulatory mechanisms are increasingly recognized as crucial determinants of cellular specification and differentiation. During muscle cell differentiation (myogenesis), extensive remodelling of histone acetylation and methylation occurs. Several of these histone modifications aid in the expression of muscle-specific genes and the silencing of genes that block lineage commitment. Therefore, the identification of new epigenetic regulatory mechanisms is of high interest. Still, the functional relevance of numerous histone modifications during myogenesis remain completely uncertain. In this study, we focus on the function of H3K36me3 and its epigenetic writer, SET domain containing 2 (SETD2), in the context of muscle cell differentiation. We first observed that SETD2 expression increases during myogenesis. Targeted depletion of SETD2 in undifferentiated (myoblasts) and differentiated (myotubes) muscle cells reduced H3K36me3 levels and induced profound changes in gene expression and slight alterations in alternative splicing, as determined by deep RNA-sequencing analysis. Enzymes that function in metabolic pathways were upregulated in response to SETD2 depletion. Furthermore, we demonstrated that upregulation of several glycolytic enzymes was associated with an increase in intracellular pyruvate levels in SETD2-depleted cells, indicating a novel role for SETD2 in metabolic programming during myogenesis. Together, our results provide new insight into the signalling pathways controlled by chromatin-modifying enzymes and their associated histone modifications during muscle cell differentiation.

Stretching muscle cells induces transcriptional and splicing transitions and changes in SR proteins.

Alternative splicing is an RNA processing mechanism involved in skeletal muscle development and pathology. Muscular diseases exhibit splicing alterations and changes in mechanobiology leading us to investigate the interconnection between mechanical forces and RNA processing. We performed deep RNA-sequencing after stretching muscle cells. First, we uncovered transcriptional changes in genes encoding proteins involved in muscle function and transcription. Second, we observed that numerous mechanosensitive genes were part of the MAPK pathway which was activated in response to stretching. Third, we revealed that stretching skeletal muscle cells increased the proportion of alternatively spliced cassette exons and their inclusion. Fourth, we demonstrated that the serine and arginine-rich proteins exhibited stronger transcriptional changes than other RNA-binding proteins and that SRSF4 phosphorylation is mechanosensitive. Identifying SRSF4 as a mechanosensitive RNA-binding protein that might contribute to crosstalk between mechanotransduction, transcription, and splicing could potentially reveal novel insights into muscular diseases, particularly those with unknown etiologies.

It's not just a phase: function and characteristics of RNA-binding proteins in phase separation.

Biomolecular condensates that form via phase separation are increasingly regarded as coordinators of cellular reactions that regulate a wide variety of biological phenomena. Mounting evidence suggests that multiple steps of the RNA life cycle are organized within RNA-binding protein-rich condensates. In this Review, we discuss recent insights into the influence of phase separation on RNA biology, which has implications for basic cell biology, the pathogenesis of human diseases and the development of novel therapies.

ViaFuse: Fiji macros to calculate skeletal muscle cell viability and fusion index.

BACKGROUND: Measuring biological features of skeletal muscle cells is difficult because of their unique morphology and multinucleate nature upon differentiation. Here, we developed a new Fiji macro package called ViaFuse (that stands for viability and fusion) to measure skeletal muscle cell viability and differentiation. To test ViaFuse, we utilized immunofluorescence images of differentiated myotubes where the capping actin protein of muscle z-line subunit beta (CAPZB) was depleted in comparison with control cells. RESULTS: We compared the values achieved using the ViaFuse macros first with manual quantification performed by researchers and second with those obtained utilizing the MATLAB muscle-centric software MyoCount. We observed a high degree of correlation between all methods of quantification. CONCLUSIONS: ViaFuse can detect the borders of myotubes and identify nuclear clumps which have been limitations of previous muscle-centric imaging software. The ViaFuse macros require little computer power or space to run and user inputs to the ViaFuse macros are minimal, thereby automating the analysis process in a quick, easy, and accurate fashion. Additionally, the ViaFuse macros work with Fiji, an existing imaging software widely used by skeletal muscle researchers. Furthermore, ViaFuse is compatible with many computer systems, has a very intuitive interface, and does not require prior complex mathematical knowledge. Therefore, we propose ViaFuse as a robust and meticulous method to quantify skeletal muscle cell viability and differentiation.

CRTC1/MAML2 directs a PGC-1α-IGF-1 circuit that confers vulnerability to PPARγ inhibition.

Mucoepidermoid carcinoma (MEC) is a life-threatening salivary gland cancer that is driven primarily by a transcriptional coactivator fusion composed of cyclic AMP-regulated transcriptional coactivator 1 (CRTC1) and mastermind-like 2 (MAML2). The mechanisms by which the chimeric CRTC1/MAML2 (C1/M2) oncoprotein rewires gene expression programs that promote tumorigenesis remain poorly understood. Here, we show that C1/M2 induces transcriptional activation of the non-canonical peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) splice variant PGC-1α4, which regulates peroxisome proliferator-activated receptor gamma (PPARγ)-mediated insulin-like growth factor 1 (IGF-1) expression. This mitogenic transcriptional circuitry is consistent across cell lines and primary tumors. C1/M2-positive tumors exhibit IGF-1 pathway activation, and small-molecule drug screens reveal that tumor cells harboring the fusion gene are selectively sensitive to IGF-1 receptor (IGF-1R) inhibition. Furthermore, this dependence on autocrine regulation of IGF-1 transcription renders MEC cells susceptible to PPARγ inhibition with inverse agonists. These results yield insights into the aberrant coregulatory functions of C1/M2 and identify a specific vulnerability that can be exploited for precision therapy.

FXR1 splicing is important for muscle development and biomolecular condensates in muscle cells.

Fragile-X mental retardation autosomal homologue-1 (FXR1) is a muscle-enriched RNA-binding protein. FXR1 depletion is perinatally lethal in mice, Xenopus, and zebrafish; however, the mechanisms driving these phenotypes remain unclear. The FXR1 gene undergoes alternative splicing, producing multiple protein isoforms and mis-splicing has been implicated in disease. Furthermore, mutations that cause frameshifts in muscle-specific isoforms result in congenital multi-minicore myopathy. We observed that FXR1 alternative splicing is pronounced in the serine- and arginine-rich intrinsically disordered domain; these domains are known to promote biomolecular condensation. Here, we show that tissue-specific splicing of fxr1 is required for Xenopus development and alters the disordered domain of FXR1. FXR1 isoforms vary in the formation of RNA-dependent biomolecular condensates in cells and in vitro. This work shows that regulation of tissue-specific splicing can influence FXR1 condensates in muscle development and how mis-splicing promotes disease.