scholarly journals The SWI/SNF subunits BRG1 affects alternative splicing by changing RNA binding factor interactions with RNA

2019 ◽  
Author(s):  
Antoni Gañez Zapater ◽  
Sebastian D. Mackowiak ◽  
Yuan Guo ◽  
Antonio Jordan-Pla ◽  
Marc R. Friedländer ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
Nucleosome Position ◽  
Gc Content ◽  
Chromatin Remodelling ◽  
Transcription Rate ◽  
Interaction Patterns ◽  
Exon Inclusion ◽  
Regulatory Factors ◽  
Binding Factor

AbstractBRG1 and BRM are ATPase core subunits of the human SWI/SNF chromatin remodelling complexes. The function of the SWI/SNF complexes in transcriptional initiation has been well studied, while a function in alternative splicing has only been studied for a few cases for BRM-containing SWI/SNF complexes. Here, we have expressed BRG1 in C33A cells, a BRG1 and BRM-deficient cell line, and we have analysed the effects on the transcriptome by RNA sequencing. We have shown that BRG1 expression affects the splicing of a subset of genes. For some, BRG1 expression favours exon inclusion and for others, exon skipping. Some of the changes in alternative splicing induced by BRG1 expression do not require the ATPase activity of BRG1. Among the exons regulated through an ATPase-independent mechanism, the included exons had signatures of high GC-content and lacked a positioned nucleosome at the exon. By investigating three genes in which the expression of either wild-type BRG1 or a BRG1-ATPase-deficient variant favoured exon inclusion, we showed that expression of the ATPases promotes the local recruitment of RNA binding factors to chromatin and RNA in a differential manner. The hnRNPL, hnRNPU and SAM68 proteins associated to chromatin in C33A cells expressing BRG1 or BRM, but their association with RNA varied. We propose that SWI/SNF can regulate alternative splicing by interacting with splicing-RNA binding factor and altering their binding to the nascent pre-mRNA, which changes RNP structure.Author summarySplicing, in particular alternative splicing, is a combinatorial process which involves splicing factor complexes and many RNA binding splicing regulatory proteins in different constellations. Most splicing events occur during transcription, which also makes the DNA sequence, the chromatin state and the transcription rate at the exons important components that influence the splicing outcome. We show here that the ATP-dependent chromatin remodelling complex SWI/SNF influences the interactions of splicing regulatory factors with RNA during transcription on certain exons that have a high GC-content. The splicing on this type of exon rely on the ATPase BRG1 and favour inclusion of alternative exons in an ATP-independent manner. SWI/SNF complexes are known to alter the chromatin structure at promoters in transcription initiation, and have been previously shown to alter the transcription rate or nucleosome position in splicing. Our results suggests a further mechanism for chromatin remodelling proteins in splicing: to change the interaction patterns of RNA binding splicing regulatory factors at alternative exons to alter the splicing outcome.

scholarly journals The CELF Family of RNA Binding Proteins Is Implicated in Cell-Specific and Developmentally Regulated Alternative Splicing

2001 ◽  
Vol 21 (4) ◽  
pp. 1285-1296 ◽  
Author(s):  
Andrea N. Ladd ◽  
Nicolas Charlet-B. ◽  
Thomas A. Cooper
Keyword(s):  
Alternative Splicing ◽  
Heart Development ◽  
Binding Proteins ◽  
Rna Binding ◽  
Striated Muscle ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Protein Isoforms ◽  
Developmentally Regulated ◽  
Exon Inclusion

ABSTRACT Alternative splicing of cardiac troponin T (cTNT) exon 5 undergoes a developmentally regulated switch such that exon inclusion predominates in embryonic, but not adult, striated muscle. We previously described four muscle-specific splicing enhancers (MSEs) within introns flanking exon 5 in chicken cTNT that are both necessary and sufficient for exon inclusion in embryonic muscle. We also demonstrated that CUG-binding protein (CUG-BP) binds a conserved CUG motif within a human cTNT MSE and positively regulates MSE-dependent exon inclusion. Here we report that CUG-BP is one of a novel family of developmentally regulated RNA binding proteins that includes embryonically lethal abnormal vision-type RNA binding protein 3 (ETR-3). This family, which we call CELF proteins for CUG-BP- and ETR-3-like factors, specifically bound MSE-containing RNAs in vitro and activated MSE-dependent exon inclusion of cTNT minigenes in vivo. The expression of two CELF proteins is highly restricted to brain. CUG-BP, ETR-3, and CELF4 are more broadly expressed, and expression is developmentally regulated in striated muscle and brain. Changes in the level of expression and isoforms of ETR-3 in two different developmental systems correlated with regulated changes in cTNT splicing. A switch from cTNT exon skipping to inclusion tightly correlated with induction of ETR-3 protein expression during differentiation of C2C12 myoblasts. During heart development, the switch in cTNT splicing correlated with a transition in ETR-3 protein isoforms. We propose that ETR-3 is a major regulator of cTNT alternative splicing and that the CELF family plays an important regulatory role in cell-specific alternative splicing during normal development and disease.

scholarly journals Nuclear matrix-associated protein SMAR1 regulates alternative splicing via HDAC6-mediated deacetylation of Sam68

2015 ◽  
Vol 112 (26) ◽  
pp. E3374-E3383 ◽  
Author(s):  
Kiran Kumar Nakka ◽  
Nidhi Chaudhary ◽  
Shruti Joshi ◽  
Jyotsna Bhat ◽  
Kulwant Singh ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Alternative Splicing ◽  
Posttranslational Modifications ◽  
Rna Binding ◽  
Mapk Pathway ◽  
Binding Protein ◽  
Histone Deacetylase 6 ◽  
Exon Inclusion ◽  
Variant Exon

Pre-mRNA splicing is a complex regulatory nexus modulated by various trans-factors and their posttranslational modifications to create a dynamic transcriptome through alternative splicing. Signal-induced phosphorylation and dephosphorylation of trans-factors are known to regulate alternative splicing. However, the role of other posttranslational modifications, such as deacetylation/acetylation, methylation, and ubiquitination, that could modulate alternative splicing in either a signal-dependent or -independent manner remain enigmatic. Here, we demonstrate that Scaffold/matrix-associated region-binding protein 1 (SMAR1) negatively regulates alternative splicing through histone deacetylase 6 (HDAC6)-mediated deacetylation of RNA-binding protein Sam68 (Src-associated substrate during mitosis of 68 kDa). SMAR1 is enriched in nuclear splicing speckles and associates with the snRNAs that are involved in splice site recognition. ERK–MAPK pathway that regulates alternative splicing facilitates ERK-1/2–mediated phosphorylation of SMAR1 at threonines 345 and 360 and localizes SMAR1 to the cytoplasm, preventing its interaction with Sam68. We showed that endogenously, SMAR1 through HDAC6 maintains Sam68 in a deacetylated state. However, knockdown or ERK-mediated phosphorylation of SMAR1 releases the inhibitory SMAR1–HDAC6–Sam68 complex, facilitating Sam68 acetylation and alternative splicing. Furthermore, loss of heterozygosity at the Chr.16q24.3 locus in breast cancer cells, wherein the human homolog of SMAR1 (BANP) has been mapped, enhances Sam68 acetylation and CD44 variant exon inclusion. In addition, tail-vein injections in mice with human breast cancer MCF-7 cells depleted for SMAR1 showed increased CD44 variant exon inclusion and concomitant metastatic propensity, confirming the functional role of SMAR1 in regulation of alternative splicing. Thus, our results reveal the complex molecular mechanism underlying SMAR1-mediated signal-dependent and -independent regulation of alternative splicing via Sam68 deacetylation.

scholarly journals The HIV 5′ Gag Region Displays a Specific Nucleotide Bias Regulating Viral Splicing and Infectivity

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 997
Author(s):  
Bastian Grewe ◽  
Carolin Vogt ◽  
Theresa Horstkötter ◽  
Bettina Tippler ◽  
Han Xiao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Nuclear Export ◽  
Rna Binding ◽  
Gc Content ◽  
Splice Donor Site ◽  
Viral Gene ◽  
Nucleotide Bias ◽  
Rna Sequence ◽  
Hiv Rna

Alternative splicing and the expression of intron-containing mRNAs is one hallmark of HIV gene expression. To facilitate the otherwise hampered nuclear export of non-fully processed mRNAs, HIV encodes the Rev protein, which recognizes its intronic response element and fuels the HIV RNAs into the CRM-1-dependent nuclear protein export pathway. Both alternative splicing and Rev-dependency are regulated by the primary HIV RNA sequence. Here, we show that these processes are extremely sensitive to sequence alterations in the 5’coding region of the HIV genomic RNA. Increasing the GC content by insertion of either GFP or silent mutations activates a cryptic splice donor site in gag, entirely deregulates the viral splicing pattern, and lowers infectivity. Interestingly, an adaptation of the inserted GFP sequence toward an HIV-like nucleotide bias reversed these phenotypes completely. Of note, the adaptation yielded completely different primary sequences although encoding the same amino acids. Thus, the phenotypes solely depend on the nucleotide composition of the two GFP versions. This is a strong indication of an HIV-specific mRNP code in the 5′ gag region wherein the primary RNA sequence bias creates motifs for RNA-binding proteins and controls the fate of the HIV-RNA in terms of viral gene expression and infectivity.

scholarly journals Srrm234, but not canonical SR and hnRNP proteins drive inclusion of Dscam exon 9 variable exons

2019 ◽  
Author(s):  
Pinar Ustaoglu ◽  
Irmgard U. Haussmann ◽  
Hongzhi Liao ◽  
Antonio Torres-Mendez ◽  
Roland Arnold ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Alternative Splicing ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Main Role ◽  
Sr Protein ◽  
Exon Inclusion ◽  
Hnrnp Proteins ◽  
Exon 9

AbstractAlternative splicing of pre-mRNA is a major mechanism to diversify protein functionality in metazoans from a limited number of genes. In the Drosophila melanogaster Down Syndrome Cell Adhesion Molecule (Dscam) important for neuronal wiring up to 38,016 isoforms can be generated by mutually exclusive alternative splicing in four clusters of variable exons. However, it is not understood how a specific exon is chosen from the many variables and how variable exons are prevented from being spliced together. A main role in the regulation of Dscam alternative splicing has been attributed to RNA binding proteins, but how they impact on exon selection is not well understood. Serine-arginine-rich (SR) proteins and hnRNP proteins are the two main types of RNA binding proteins with major roles in exon definition and splice site selection. Here, we analyzed the role of SR and hnRNP proteins in Dscam exon 9 alternative splicing in mutant Drosophila melanogaster embryos because of their essential function for development. Strikingly, loss or overexpression of canonical SR and hnRNP proteins even when multiple proteins are depleted together, does not affect Dscam alternative exon selection very dramatically. Conversely, non-canonical SR protein Serine-arginine repetitive matrix 2/3/4 (Srrm234) is a main determinant of exon inclusion in Dscam exon 9 cluster. Since long-range base-pairings are absent in the exon 9 cluster, our data argue for a small complement of regulatory factors as main determinants of exon inclusion in the Dscam exon 9 cluster.

scholarly journals PTBP1 promotes cardiac hypertrophy and diastolic dysfunction by modulating alternative splicing

2020 ◽  
Author(s):  
Carlos Martí-Gómez ◽  
Javier Larrasa-Alonso ◽  
Marina López-Olañeta ◽  
María Villalba-Orero ◽  
Pablo García-Pavía ◽  
...  
Keyword(s):  
Heart Disease ◽  
Alternative Splicing ◽  
Cardiac Hypertrophy ◽  
Diastolic Dysfunction ◽  
Protein Interaction ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mouse Heart ◽  
Interaction Patterns

Alternative splicing (AS) plays a major role in the generation of transcript diversity. In the heart, roles have been described for some AS variants and individual regulatory RNA binding proteins (RBPs); however, the global impact and regulation of AS patterns in cardiac pathophysiology is poorly understood. Here, we studied the AS profiles in heart disease, their relationship with heart development and the regulatory mechanisms control-ling AS dynamics in the mouse heart using a total of 136 RNA-seq samples. We found that AS and gene expression changes affect different genes, which are also involved in distinct biological functions. Developmental AS changes were more abundant and had stronger predicted impact on the encoded protein than those taking place during heart disease. However, AS changes in heart disease significantly modified protein interaction patterns and rewire the protein-protein interaction network. Using a database of experimentally determined binding sites of a large collection of RNA binding proteins, we studied the regulatory proteins associated to AS changes in each condition. Computational modelling revealed that developmental transitions were mainly driven by the up-regulation of MBNL1, whereas disease associated AS changes were driven by a more complex regulatory network, characterized by the interaction of different RNA binding proteins, with PTBP1 as the largest individual modulator. In adult mice, PTBP1 over-expression was sufficient to induce cardiac hypertrophy and diastolic dysfunction and significantly alter the AS profile. Overall, our study provides new in-sights into the functional impact of AS patterns in cardiac physiology and how computationally driven hypotheses can help to improve our understanding of RNA regulation and its contribution to heart disease.

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

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.

scholarly journals A 5′-tRNA halve, tiRNA-Gly promotes cell proliferation and migration via binding to RBM17 and inducing alternative splicing in papillary thyroid cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Litao Han ◽  
Hejing Lai ◽  
Yichen Yang ◽  
Jiaqian Hu ◽  
Zhe Li ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Thyroid Cancer ◽  
Alternative Splicing ◽  
Papillary Thyroid Cancer ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Papillary Thyroid ◽  
Cell Proliferation And Migration ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background tRNA-derived small noncoding RNAs (sncRNAs) are mainly categorized into tRNA halves (tiRNAs) and fragments (tRFs). Biological functions of tiRNAs in human solid tumor are attracting more and more attention, but researches concerning the mechanisms in tiRNAs-mediated tumorigenesis are rarely. The direct regulatory relationship between tiRNAs and splicing-related proteins remain elusive. Methods Papillary thyroid carcinoma (PTC) associated tRNA fragments were screened by tRNA fragments deep sequencing and validated by qRT-PCR and Northern Blot in PTC tissues. The biological function of tRNA fragments were assessed by cell counting kit, transwells and subcutaneous transplantation tumor of nude mice. For mechanistic study, tRNA fragments pull-down, RNA immunoprecipitation, Western Blot, Immunofluorescence, Immunohistochemical staining were performed. Results Herein, we have identified a 33 nt tiRNA-Gly significantly increases in papillary thyroid cancer (PTC) based on tRFs & tiRNAs sequencing. The ectopic expression of tiRNA-Gly promotes cell proliferation and migration, whereas down-regulation of tiRNA-Gly exhibits reverse effects. Mechanistic investigations reveal tiRNA-Gly directly bind the UHM domain of a splicing-related RNA-binding protein RBM17. The interaction with tiRNA-Gly could translocate RBM17 from cytoplasm into nucleus. In addition, tiRNA-Gly increases RBM17 protein expression via inhibiting its degradation in a ubiquitin/proteasome-dependent way. Moreover, RBM17 level in tiRNA-Gly high-expressing human PTC tissues is upregulated. In vivo mouse model shows that suppression of tiRNA-Gly decreases RBM17 expression. Importantly, tiRNA-Gly can induce exon 16 splicing of MAP4K4 mRNA leading to phosphorylation of downstream signaling pathway, which is RBM17 dependent. Conclusions Our study firstly illustrates tiRNA-Gly can directly bind to RBM17 and display oncogenic effect via RBM17-mediated alternative splicing. This fully novel model broadens our understanding of molecular mechanism in which tRNA fragment in tumor cells directly bind RNA binding protein and play a role in alternative splicing.

scholarly journals RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing

Oncogene ◽  
2021 ◽  
Author(s):  
Qiuxia Yan ◽  
Peng Zeng ◽  
Xiuqin Zhou ◽  
Xiaoying Zhao ◽  
Runqiang Chen ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Aerobic Glycolysis ◽  
Histological Grade ◽  
Migration And Invasion ◽  
Binding Motif ◽  
Hnrnp A1

AbstractThe prognosis for patients with metastatic bladder cancer (BCa) is poor, and it is not improved by current treatments. RNA-binding motif protein X-linked (RBMX) are involved in the regulation of the malignant progression of various tumors. However, the role of RBMX in BCa tumorigenicity and progression remains unclear. In this study, we found that RBMX was significantly downregulated in BCa tissues, especially in muscle-invasive BCa tissues. RBMX expression was negatively correlated with tumor stage, histological grade and poor patient prognosis. Functional assays demonstrated that RBMX inhibited BCa cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Mechanistic investigations revealed that hnRNP A1 was an RBMX-binding protein. RBMX competitively inhibited the combination of the RGG motif in hnRNP A1 and the sequences flanking PKM exon 9, leading to the formation of lower PKM2 and higher PKM1 levels, which attenuated the tumorigenicity and progression of BCa. Moreover, RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM alternative splicing and counteracted the PKM2 overexpression-induced aggressive phenotype of the BCa cells. In conclusion, our findings indicate that RBMX suppresses BCa tumorigenicity and progression via an hnRNP A1-mediated PKM alternative splicing mechanism. RBMX may serve as a novel prognostic biomarker for clinical intervention in BCa.

scholarly journals Targeting Poison Exons to Treat Developmental and Epileptic Encephalopathy

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.

scholarly journals Nova Regulates GABAA Receptor γ2 Alternative Splicing via a Distal Downstream UCAU-Rich Intronic Splicing Enhancer

2003 ◽  
Vol 23 (13) ◽  
pp. 4687-4700 ◽  
Author(s):  
B. Kate Dredge ◽  
Robert B. Darnell
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Splicing Regulation ◽  
Globin Genes ◽  
Enhancer Element ◽  
Intronic Splicing Enhancer ◽  
Splicing Enhancer

ABSTRACT Nova is a neuron-specific RNA binding protein targeted in patients with the autoimmune disorder paraneoplastic opsoclonus-myoclonus ataxia, which is characterized by failure of inhibition of brainstem and spinal motor systems. Here, we have biochemically confirmed the observation that splicing regulation of the inhibitory GABAA receptor γ2 (GABAARγ2) subunit pre-mRNA exon E9 is disrupted in mice lacking Nova-1. To elucidate the mechanism by which Nova-1 regulates GABAARγ2 alternative splicing, we systematically screened minigenes derived from the GABAARγ2 and human β-globin genes for their ability to support Nova-dependent splicing in transient transfection assays. These studies demonstrate that Nova-1 acts directly on GABAARγ2 pre-mRNA to regulate E9 splicing and identify an intronic region that is necessary and sufficient for Nova-dependent enhancement of exon inclusion, which we term the NISE (Nova-dependent intronic splicing enhancer) element. The NISE element (located 80 nucleotides upstream of the splice acceptor site of the downstream exon E10) is composed of repeats of the sequence YCAY, consistent with previous studies of the mechanism by which Nova binds RNA. Mutation of these repeats abolishes binding of Nova-1 to the RNA in vitro and Nova-dependent splicing regulation in vivo. These data provide a molecular basis for understanding Nova regulation of GABAARγ2 alternative splicing and suggest that general dysregulation of Nova's splicing enhancer function may underlie the neurologic defects seen in Nova's absence.

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