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Alternative splicing results in disordered protein isoforms that are capable of phase separation. (A) Disorder predictions based on the primary aa sequence of FXR1 isoforms A and E using the IUPRED algorithm. (B) Endogenous FXR1 protein was visualized by immunofluorescence in myotubes using an antibody against the N terminus of FXR1 (recognizes all splice variants). Representative images depicting FXR1 puncta. Scale bar, 5 µm. Inset shows droplets magnified fourfold. Dapi, 6′-diamidino-2-phenylindole. (C) Time-lapse acquisition of human isoform 2 FXR1-EGFP (pAGB828). Two condensates (red and blue arrows) fuse together to form one (purple arrow). Scale bar, 1 µm. (D) C2C12 myoblasts were differentiated for 1, 2, or 3 d (D1, D2, D3). Cell lysates and pellets were analyzed by Western blot using an FXR1 antibody specific to the C terminus of isoform E. (E–G) MOs targeting exons 15 (mo-1) and/or 16 (mo-2) were delivered in myoblasts, and the next day cells were differentiated for 4 d. Cells were analyzed by Western blotting and immunofluorescence using an antibody against FXR1 N terminus (E). The CV was calculated as a measurement of FXR1 condensation. P > 0.5 mo-1 versus mo-control, P < 0.001 mo-2 and mo-1 & 2 versus mo-control (F). Representative images of MO-treated cells showing FXR1 puncta. Scale bar, 5 µm. Bottom row shows droplets magnified fivefold (G). Data are standard box and whisker plot. Fig. 4 is linked to Fig. S3, F–I.
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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 implica...
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Neurons critically rely on the functions of RNA-binding proteins to maintain their polarity and resistance to neurotoxic stress. HnRNP R has a diverse range of post-transcriptional regulatory functions and is important for neuronal development by regulating axon growth. Hnrnpr pre-mRNA undergoes alternative splicing giving rise to a full-length pro...
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... Alteration of the mRNA nucleotide sequence could affect protein splicing. For example, tissue-specific splicing of FXR1 modifies its disordered region thus affecting its ability to phase separate in cells and in vitro [158]. Post-transcriptional modifications of mRNA sequence (e.g. ...
The term "biomolecular condensates" is used to describe membraneless compartments in eukaryotic cells, accumulating proteins and nucleic acids. Biomolecular condensates are formed as a result of liquid-liquid phase separation (LLPS). Often, they demonstrate properties of liquid-like droplets or gel-like aggregates; however, some of them may appear to have a more complex structure and high-order organization. Membraneless micro-compartments are involved in diverse processes both in cytoplasm and in nucleus, among them ribosome biogen-esis, regulation of gene expression, cell signaling, and stress response. Condensates properties and structure could be highly dynamic and are affected by various internal and external factors, e.g., concentration and interactions of components, solution temperature, pH, osmolarity, etc. In this review, we discuss variety of biomolecular conden-sates and their functions in live cells, describe their structure variants, highlight domain and primary sequence organization of the constituent proteins and nucleic acids. Finally, we describe current advances in methods that characterize structure, properties, morphology, and dynamics of biomolecular condensates in vitro and in vivo.
... Neonatal (PN4.5) and adult (4-month-old) mice were euthanized by isoflurane inhalant followed by secondary physical euthanasia. 68 Harvested tissues were immediately snap frozen. Three-to four-month-old wild-type C57BL/6J male mice were subjected to transverse aortic constriction or sham surgeries followed by serial echocardiography prior to euthanasia (see Transthoracic echocardiography). ...
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.
... Alternative splicing of the FXR1 pre-mRNA generates a variety of FXR1 protein isoforms in murine and human cells (19,29). Since the FXR1 isoform nomenclature is perplexing, we refer to the two human FXR1 proteins studied in the present report as the short FXR1 (s-FXR1) and long FXR1 (l-FXR1) based on the length of their C-terminal intrinsically disordered domain (IDD) (see below). ...
... The s-FXR1 protein forms distinct subcellular condensates in HAdV-5-infected cells. A recent study has shown that mouse FXR1 isoforms exhibit altered subcellular localization depending on the length of their IDD (29). To test the subcellular localization of the human FXR1 isoforms, immunofluorescence experiments were performed both in lentivirus-transduced A549 FXR1-KO and in plasmid-transfected HeLa cells. ...
... FXR1 is an RNA-binding protein that regulates mRNA localization, stability, and translation (22-25, 27, 44). Moreover, FXR1 controls muscle and neuron development and has a potential oncogenic role in many cancers (17,29,45). Despite a detailed knowledge of the role of FXR1 in cellular gene expression, very little is known about its exact functions in virus infections (46,47). ...
Human adenoviruses (HAdVs) are widespread pathogens causing a variety of diseases. A well-controlled expression of virus capsid mRNAs originating from the major late transcription unit (MLTU) is essential for forming the infectious virus progeny. However, regulation of the MLTU mRNA metabolism has mainly remained enigmatic. In this study, we show that the cellular RNA-binding protein FXR1 controls the stability of the HAdV-5 MLTU mRNAs, as depletion of FXR1 resulted in increased steady-state levels of MLTU mRNAs. Surprisingly, the lack of FXR1 reduced viral capsid protein accumulation and formation of the infectious virus progeny, indicating an opposing function of FXR1 in HAdV-5 infection. Further, the long FXR1 isoform interfered with MLTU mRNA translation, suggesting FXR1 isoform-specific functions in virus-infected cells. We also show that the FXR1 protein interacts with N6-methyladenosine (m6A)-modified MLTU mRNAs, thereby acting as a novel m6A reader protein in HAdV-5 infected cells. Collectively, our study identifies FXR1 as a regulator of MLTU mRNA metabolism in the lytic HAdV-5 life cycle. IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing various self-limiting diseases, such as the common cold and conjunctivitis. Even though adenoviruses have been studied for more than 6 decades, there are still gaps in understanding how the virus interferes with the host cell to achieve efficient growth. In this study, we identified the cellular RNA-binding protein FXR1 as a factor manipulating the HAdV life cycle. We show that the FXR1 protein specifically interferes with mRNAs encoding essential viral capsid proteins. Since the lack of the FXR1 protein reduces virus growth, we propose that FXR1 can be considered a novel cellular proviral factor needed for efficient HAdV growth. Collectively, our study provides new detailed insights about the HAdV-host interactions, which might be helpful when developing countermeasures against pathogenic adenovirus infections and for improving adenovirus-based therapies.
... When the normal splicing is disrupted, missplicing causes functionally altered proteins, resulting in the occurrence of human diseases [4]. Conditions linked to RNA splicing defects mainly include cancer, cardiac dysplasia, muscular dystrophy, and neurodegenerative diseases such as Parkinson's and Huntington's disease [5][6][7][8][9]. For instance, nearly 50 % of individuals with myelodysplastic syndromes (MDS) have mutations in SF3B1, SRSF2, or U2AF1 splicing factors, which impair hematopoiesis and promote the growth of acute myeloid leukemia (AML) [10,11]. ...
The RNA splicing process which removes introns from nascent transcripts is an indispensable step in gene expression. The life processes of organisms are composed of a range of different mRNA variants that are translated into proteins with various functions as a result of alternative splicing. Monitoring and controlling RNA splicing can successfully repair the dangerous mutant genes that underlie various diseases. However, attempts to uncover specific elements in the regulation of splicing are hampered by the absence of appropriate tools. Traditional RNA splicing detection technology frequently focuses on the identification and analysis of post-splicing products, which is often accompanied by irreversible damage to the detected objects. It cannot provide dynamic and detailed descriptions of regulatory factors, functional elements, and spatiotemporal distributions during the splicing process. It is still difficult to identify and measure aberrant RNA splicing in living cells and in vivo. New technical tools have sprung up, providing fresh motivation and guidance for the identification of RNA splicing. Here, based on the genetically encoded reporter gene system, we cover in detail the monitoring, imaging and biomedical applications of RNA splicing process using different types of reporter gene systems.
... FXR1 is required for muscle development in mice (41). Similar to our observations, a recent study showed that FXR1 also formed biomolecular condensates in muscle cells (42), but the function of FXR1 phase separation in translational control and muscle development remains to be determined. Recessive mutations in exon 15 of FXR1 are associated with congenital multi-minicore myopathy, and a patient carrying such mutations also showed hypoplastic genitalia and cryptorchidism (43). ...
Postmeiotic spermatids use a unique strategy to coordinate gene expression with morphological transformation, in which transcription and translation take place at separate developmental stages, but how mRNAs stored as translationally inert messenger ribonucleoproteins in developing spermatids become activated remains largely unknown. Here, we report that the RNA binding protein FXR1, a member of the fragile X-related (FXR) family, is highly expressed in late spermatids and undergoes liquid-liquid phase separation (LLPS) to merge messenger ribonucleoprotein granules with the translation machinery to convert stored mRNAs into a translationally activated state. Germline-specific Fxr1 ablation in mice impaired the translation of target mRNAs and caused defective spermatid development and male infertility, and a phase separation-deficient FXR1L351P mutation in Fxr1 knock-in mice produced the same developmental defect. These findings uncover a mechanism for translational reprogramming with LLPS as a key driver in spermiogenesis.
... As L-bodies, like many other biomolecular condensates, are highly enriched for proteins with multiple RNA binding domains and IDRs, the relative contribution of each of these types of interactions to the dynamics of the condensate is an important outstanding question. For example, in FXR1 assemblies, RNA binding drives phase separation and IDRs of various lengths tune the dynamics of the condensate (Smith et al., 2020). Similarly, in L-bodies, IDR-containing proteins, which are more dynamic than PTBP3 , may function to keep the condensate in a more liquid-like state, perhaps preventing an irreversible transition to a solid-like state. ...
RNA localization and biomolecular condensate formation are key biological strategies for organizing the cytoplasm and generating cellular polarity. In Xenopus oocytes, RNAs required for germ layer patterning localize in biomolecular condensates, termed Localization bodies (L-bodies). Here, we have used an L-body RNA-binding protein, PTBP3, to test the role of RNA–protein interactions in regulating the biophysical characteristics of L-bodies in vivo and PTBP3–RNA condensates in vitro. Our results reveal that RNA–protein interactions drive recruitment of PTBP3 and localized RNA to L-bodies and that multivalent interactions tune the dynamics of the PTBP3 after localization. In a concentration dependent manner, RNA becomes non-dynamic and interactions with the RNA determine PTBP3 dynamics within these biomolecular condensates in vivo and in vitro. Importantly, RNA, but not protein, is required for maintenance of the PTBP3–RNA condensates in vitro, pointing to a model where RNA serves as a non-dynamic substructure in these condensates.
... Several cytoplasmic condensates have been found that are generated by mRNA-dependent phase separation ( Figure 1A). They include TIS granules, FXR1 condensates, and L-bodies, and they were detected in human cell lines, mouse myotubes, and in frog oocyte stages II-III, respectively (Ma and Mayr, 2018;Smith et al., 2020;Neil et al., 2021). TIS granules are generated by assembly of the RNA-binding protein TIS11B. ...
... FXR1 condensates are generated through RNA-dependent assembly of the RNA-binding protein FXR1. They were initially observed in muscle cells (Smith et al., 2020) but have also been found in human cell lines where they generate a large network in the cytoplasm, called FXR1 network (Chen and Mayr). In frog oocytes, irregularly shaped L-bodies are enriched for several RNA-binding proteins together with their bound mRNAs (Neil et al., 2021;Cabral and Mowry, 2021). ...
... What protein domains could serve as protein retention signals for cytoplasmic condensates? Diverse domains were found to contribute to the high valency of RNA-binding proteins that act as scaffolds of cytoplasmic condensates: RNA-binding domains, intrinsically disordered regions, protein interaction domains, and binding motifs for post-translational modifications (PTMs) (Zhong et al., 2000;Hofweber and Dormann, 2019;Smith et al., 2020;Yang et al., 2020;Guillen-Boixet et al., 2020). For G3BP1 it was shown that the N-terminal NTF2 domain is used for homotypic binding that is necessary for stress granule assembly. ...
Most cellular processes are carried out by protein complexes, but it is still largely unknown how the subunits of lowly expressed complexes find each other in the crowded cellular environment. Here, we will describe a working model where RNA-binding proteins in cytoplasmic condensates act as matchmakers between their bound proteins (called protein targets) and newly translated proteins of their RNA targets to promote their assembly into complexes. Different RNA-binding proteins act as scaffolds for various cytoplasmic condensates with several of them supporting translation. mRNAs and proteins are recruited into the cytoplasmic condensates through binding to specific domains in the RNA-binding proteins. Scaffold RNA-binding proteins have a high valency. In our model, they use homotypic interactions to assemble condensates and they use heterotypic interactions to recruit protein targets into the condensates. We propose that unoccupied binding sites in the scaffold RNA-binding proteins transiently retain recruited and newly translated proteins in the condensates, thus promoting their assembly into complexes. Taken together, we propose that lowly expressed subunits of protein complexes combine information in their mRNAs and proteins to colocalize in the cytoplasm. The efficiency of protein complex assembly is increased by transient entrapment accomplished by multivalent RNA-binding proteins within cytoplasmic condensates.
... However, there is evidence that FXR1P promotes myogenic differentiation in C2C12 cells and in human myoblasts by regulating the stability of p21 mRNA, thus its loss of function causes an up-regulation of p21 activity and induces a premature cell-cycle arrest that impairs myogenesis (Davidovic et al., 2013). Recently, It has been shown that the developmentally regulated muscle-specific inclusion of exon 15 in Fxr1 mRNA is required for the formation of biomolecular condensates in differentiating C2C12 myoblasts and for somite organization in Xenopus embryos (Smith et al., 2020). The property of FXR1P to undergo liquid-liquid phase separation is dependent on its intrinsically disordered domain (IDD) encoded by exon 15 and on its RNA-binding function. ...
... The property of FXR1P to undergo liquid-liquid phase separation is dependent on its intrinsically disordered domain (IDD) encoded by exon 15 and on its RNA-binding function. It is possible that these liquid-like assemblies may pattern the developing muscle by modulating FXR1P interaction with other RBPs or mRNA targets (Smith et al., 2020). Contrary to FXR1P, there is at present no functional evidence of FXR2P in muscle development. ...
Embryonic myogenesis is a temporally and spatially regulated process that generates skeletal muscle of the trunk and limbs. During this process, mononucleated myoblasts derived from myogenic progenitor cells within the somites undergo proliferation, migration and differentiation to elongate and fuse into multinucleated functional myofibers. Skeletal muscle is the most abundant tissue of the body and has the remarkable ability to self-repair by re-activating the myogenic program in muscle stem cells, known as satellite cells. Post-transcriptional regulation of gene expression mediated by RNA-binding proteins is critically required for muscle development during embryogenesis and for muscle homeostasis in the adult. Differential subcellular localization and activity of RNA-binding proteins orchestrates target gene expression at multiple levels to regulate different steps of myogenesis. Dysfunctions of these post-transcriptional regulators impair muscle development and homeostasis, but also cause defects in motor neurons or the neuromuscular junction, resulting in muscle degeneration and neuromuscular disease. Many RNA-binding proteins, such as members of the muscle blind-like (MBNL) and CUG-BP and ETR-3-like factors (CELF) families, display both overlapping and distinct targets in muscle cells. Thus they function either cooperatively or antagonistically to coordinate myoblast proliferation and differentiation. Evidence is accumulating that the dynamic interplay of their regulatory activity may control the progression of myogenic program as well as stem cell quiescence and activation. Moreover, the role of RNA-binding proteins that regulate post-transcriptional modification in the myogenic program is far less understood as compared with transcription factors involved in myogenic specification and differentiation. Here we review past achievements and recent advances in understanding the functions of RNA-binding proteins during skeletal muscle development, regeneration and disease, with the aim to identify the fundamental questions that are still open for further investigations.
... As L-bodies, like many other biomolecular condensates, are highly enriched for proteins with multiple RNA binding domains and IDRs, the relative contribution of each of these types of interactions on the dynamics of the condensate is an important outstanding question. For example, in FXR1 assemblies, RNA binding drives phase separation and IDRs of various lengths tune the dynamics of the condensate (Smith et al., 2020). Similarly, in L-bodies IDRcontaining proteins may function to keep the condensate in a more liquid-like state, perhaps preventing . ...
RNA localization and biomolecular condensate formation are key biological strategies for organizing the cytoplasm and generating cellular and developmental polarity. While enrichment of RNAs and RNA-binding proteins (RBPs) is a hallmark of both processes, the functional and structural roles of RNA-RNA and RNA-protein interactions within condensates remain unclear. Recent work from our laboratory has shown that RNAs required for germ layer patterning in Xenopus oocytes localize in novel biomolecular condensates, termed Localization bodies (L-bodies). L-bodies are composed of a non-dynamic RNA phase enmeshed in a more dynamic protein-containing phase. However, the interactions that drive the biophysical characteristics of L-bodies are not known. Here, we test the role of RNA-protein interactions using an L-body RNA-binding protein, PTBP3, which contains four RNA-binding domains (RBDs). We find that binding of RNA to PTB is required for both RNA and PTBP3 to be enriched in L-bodies in vivo. Importantly, while RNA binding to a single RBD is sufficient to drive PTBP3 localization to L-bodies, interactions between multiple RRMs and RNA tunes the dynamics of PTBP3 within L-bodies. In vitro, recombinant PTBP3 phase separates into non-dynamic structures in an RNA-dependent manner, supporting a role for RNA-protein interactions as a driver of both recruitment of components to L-bodies and the dynamics of the components after enrichment. Our results point to a model where RNA serves as a concentration-dependent, non-dynamic substructure and multivalent interactions with RNA are a key driver of protein dynamics.
... Alternative splicing of RBPs can alter the presence of domains capable of multivalent interactions, thereby affecting the phase-separation propensity of proteins 29,[40][41][42][43] (Fig. 3). For example, within the family of heterogeneous nuclear ribonucleoproteins A and D (HNRNPA and HNRNPD), five of six members have mammalian-specific alternative exons that encode IDRs 40 . ...
... Furthermore, isoform expression can change the morphology of condensates. The shortest splice variant of the RBP FMR1 autosomal homolog-1 (FXR1) forms large spherical condensates in U2OS cells, while the isoform containing a larger IDR is found in small irregularly shaped condensates 42 (Fig. 3c). This may be explained by the fact that a longer IDR presents more sites for PTMs, which can discourage condensate fusion 42 . ...
... The shortest splice variant of the RBP FMR1 autosomal homolog-1 (FXR1) forms large spherical condensates in U2OS cells, while the isoform containing a larger IDR is found in small irregularly shaped condensates 42 (Fig. 3c). This may be explained by the fact that a longer IDR presents more sites for PTMs, which can discourage condensate fusion 42 . Indeed, loss of low-complexity sequences in RBPs through alternative splicing does not always lead to a reduction 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. The organization of multiple steps of the RNA life cycle in phase-separated condensates presents a framework for understanding how sequestration of RNA-binding proteins and RNAs modulates gene expression.
