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 hnRNPH1 recruits PTBP2 and SRSF3 to cooperatively modulate alternative pre-mRNA splicing in germ cells and is essential for spermatogenesis and oogenesis

2021 ◽  
Author(s):  
Shuiqiao Yuan ◽  
Shenglei Feng ◽  
Jinmei Li ◽  
Hui Wen ◽  
Kuan Liu ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Germ Cell ◽  
Germ Cells ◽  
Rna Binding ◽  
Cell Development ◽  
Mrna Splicing ◽  
Conditional Knockout ◽  
Cell Junction ◽  
Germ Cell Development ◽  
Splicing Regulators

Abstract Coordinated regulation of alternative pre-mRNA splicing is essential for germ cell development. However, the molecular mechanism underlying that control alternative mRNA expression during germ cell development remains poorly understood. Herein, we showed that hnRNPH1, an RNA-binding protein, is highly expressed in the reproductive system and localized in the chromosomes of meiotic cells but excluded from the XY body in pachytene spermatocytes and recruits the splicing regulators PTBP2 and SRSF3 and cooperatively regulates the alternative splicing of the critical genes that are required for spermatogenesis. Conditional knockout Hnrnph1 in spermatogenic cells caused many abnormal splicing events that affect genes related to meiosis and communication between germ cells and Sertoli cells, characterized by asynapsis of chromosomes and impairment of germ-Sertoli communications, ultimately leading to male sterility. We further showed that hnRNPH1 could directly bind to SPO11 and recruit the splicing regulators PTBP2 and SRSF3 to regulate the alternative splicing of the target genes cooperatively. Strikingly, Hnrnph1 germline-specific mutant female mice were also infertile, and Hnrnph1-deficient oocytes exhibited a similar defective synapsis and cell-cell junction as shown in Hnrnph1-deficient male germ cells. Collectively, our data reveal an essential role for hnRNPH1 in regulating pre-mRNA splicing during spermatogenesis and oogenesis and support a molecular model whereby hnRNPH1 governs a network of alternative splicing events in germ cells via recruiting PTBP2 and SRSF3.

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

2017 ◽  
Vol 292 (8) ◽  
pp. 3466-3480 ◽  
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.

scholarly journals Alternative Splicing Regulates the Physiological Adaptation of the Mouse Hind Limb Postural and Phasic Muscles to Microgravity

2021 ◽  
Author(s):  
Mason Henrich ◽  
Pin Ha ◽  
John S. Adams ◽  
Chia Soo ◽  
Kang Ting ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Hind Limb ◽  
Rna Binding ◽  
Fiber Type ◽  
Differentially Expressed ◽  
Physiological Adaptation ◽  
Cross Sectional ◽  
Splicing Regulators ◽  
Physiological Adaptations ◽  
Coordinate Changes

Muscle atrophy and fiber type alterations are well-characterized physiological adaptations to microgravity with both understood to be primarily regulated by differential gene expression (DGE). While microgravity-induced DGE has been extensively investigated, adaptations to microgravity due to alternative splicing (AS) have not been studied in a mammalian model. We sought to comprehensively elucidate the transcriptomic underpinnings of microgravity-induced muscle phenotypes in mice by evaluating both DGE and changes in AS due to extended spaceflight. Tissue sections and total RNA were isolated from the gastrocnemius and quadriceps, postural and phasic muscles of the hind limb, respectively, of 32-week-old female BALB/c mice exposed to microgravity or ground control conditions for nine weeks. Immunohistochemistry disclosed muscle type-specific physiological adaptations to microgravity that included i) a pronounced reduction in muscle fiber cross-sectional area in both muscles and ii) a prominent slow-to-fast fiber type transition in the gastrocnemius. RNA sequencing revealed that DGE and AS varied across postural and phasic muscle types with preferential employment of DGE in the gastrocnemius and AS in the quadriceps. Gene ontology analysis indicated that DGE and AS regulate distinct molecular processes. Various non-differentially expressed transcripts encoding musculoskeletal proteins (Tnnt3, Tnnt1, Neb, Ryr1, and Ttn) and muscle-specific RNA binding splicing regulators (Mbnl1 and Rbfox1) were found to have significant changes in AS that altered critical functional domains of their protein products. In striking contrast, microgravity-induced differentially expressed genes were associated with lipid metabolism and mitochondrial function. Our work serves as the first comprehensive investigation of coordinate changes in DGE and AS in large limb muscles across spaceflight. We propose that substantial remodeling of pre-mRNA by AS is a major component of transcriptomic adaptation of skeletal muscle to microgravity. The alternatively spliced genes identified here could be targeted by small molecule splicing regulator therapies to address microgravity-induced changes in muscle during spaceflight.

scholarly journals Phosphorylation status of the Kep1 protein alters its affinity for its protein binding partner alternative splicing factor ASF/SF2

2006 ◽  
Vol 400 (1) ◽  
pp. 91-97 ◽  
Author(s):  
Cécile Robard ◽  
Alex Daviau ◽  
Marco Di Fruscio
Keyword(s):  
Alternative Splicing ◽  
Protein Binding ◽  
Cell Line ◽  
Topoisomerase I ◽  
Target Genes ◽  
Rna Binding ◽  
Splicing Factor ◽  
Alternatively Spliced ◽  
Alternative Splicing Factor

Mutations in the Drosophila kep1 gene, encoding a single maxi KH (K homology) domain-containing RNA-binding protein, result in a reduction of fertility in part due to the disruption of the apoptotic programme during oogenesis. This disruption is concomitant with the appearance of an alternatively spliced mRNA isoform encoding the inactive caspase dredd. We generated a Kep1 antibody and have found that the Kep1 protein is present in the nuclei of both the follicle and nurse cells during all stages of Drosophila oogenesis. We have shown that the Kep1 protein is phosphorylated in ovaries induced to undergo apoptosis following treatment with the topoisomerase I inhibitor camptothecin. We have also found that the Kep1 protein interacts specifically with the SR (serine/arginine-rich) protein family member ASF/SF2 (alternative splicing factor/splicing factor 2). This interaction is independent of the ability of Kep1 to bind RNA, but is dependent on the phosphorylation of the Kep1 protein, with the interaction between Kep1 and ASF/SF2 increasing in the presence of activated Src. Using a CD44v5 alternative splicing reporter construct, we observed 99% inclusion of the alternatively spliced exon 5 following kep1 transfection in a cell line that constitutively expresses activated Src. This modulation in splicing was not observed in the parental NIH 3T3 cell line in which we obtained 7.5% exon 5 inclusion following kep1 transfection. Our data suggest a mechanism of action in which the in vivo phosphorylation status of the Kep1 protein affects its affinity towards its protein binding partners and in turn may allow for the modulation of alternative splice site selection in Kep1–ASF/SF2-dependent target genes.

scholarly journals Long Non-Coding RNA Expression Levels Modulate Cell-Type-Specific Splicing Patterns by Altering Their Interaction Landscape with RNA-Binding Proteins

Genes ◽  
2019 ◽  
Vol 10 (8) ◽  
pp. 593 ◽  
Author(s):  
Felipe Wendt Porto ◽  
Swapna Vidhur Daulatabad ◽  
Sarath Chandra Janga
Keyword(s):  
Alternative Splicing ◽  
Cell Lines ◽  
Rna Binding ◽  
Cell Type ◽  
Specific Manner ◽  
Alternatively Spliced ◽  
A Cell ◽  
Specific Alternative ◽  
Cell Type Specific ◽  
Rna Interaction

Recent developments in our understanding of the interactions between long non-coding RNAs (lncRNAs) and cellular components have improved treatment approaches for various human diseases including cancer, vascular diseases, and neurological diseases. Although investigation of specific lncRNAs revealed their role in the metabolism of cellular RNA, our understanding of their contribution to post-transcriptional regulation is relatively limited. In this study, we explore the role of lncRNAs in modulating alternative splicing and their impact on downstream protein–RNA interaction networks. Analysis of alternative splicing events across 39 lncRNA knockdown and wildtype RNA-sequencing datasets from three human cell lines—HeLa (cervical cancer), K562 (myeloid leukemia), and U87 (glioblastoma)—resulted in the high-confidence (false discovery rate (fdr) < 0.01) identification of 11,630 skipped exon events and 5895 retained intron events, implicating 759 genes to be impacted at the post-transcriptional level due to the loss of lncRNAs. We observed that a majority of the alternatively spliced genes in a lncRNA knockdown were specific to the cell type. In tandem, the functions annotated to the genes affected by alternative splicing across each lncRNA knockdown also displayed cell-type specificity. To understand the mechanism behind this cell-type-specific alternative splicing pattern, we analyzed RNA-binding protein (RBP)–RNA interaction profiles across the spliced regions in order to observe cell-type-specific alternative splice event RBP binding preference. Despite limited RBP binding data across cell lines, alternatively spliced events detected in lncRNA perturbation experiments were associated with RBPs binding in proximal intron–exon junctions in a cell-type-specific manner. The cellular functions affected by alternative splicing were also affected in a cell-type-specific manner. Based on the RBP binding profiles in HeLa and K562 cells, we hypothesize that several lncRNAs are likely to exhibit a sponge effect in disease contexts, resulting in the functional disruption of RBPs and their downstream functions. We propose that such lncRNA sponges can extensively rewire post-transcriptional gene regulatory networks by altering the protein–RNA interaction landscape in a cell-type-specific manner.

scholarly journals Definition of germ cell lineage alternative splicing programs reveals a critical role for Quaking in specifying cardiac cell fate

2020 ◽  
Author(s):  
W. Samuel Fagg ◽  
Naiyou Liu ◽  
Ulrich Braunschweig ◽  
Xiaoting Chen ◽  
Steven G. Widen ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cell Fate ◽  
Rna Binding ◽  
Cardiac Development ◽  
Splice Variants ◽  
Critical Role ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Definitive Endoderm ◽  
Cardiac Mesoderm

SummaryAlternative splicing is critical for animal ontogeny; however, its role in the earliest developmental decision, the specification of the three embryonic germ layers, is poorly understood. By performing RNA-Seq on human embryonic stem cells (hESCs) and derived definitive endoderm, cardiac mesoderm, and ectoderm cell lineages, we detect distinct alternative splicing programs associated with each lineage, with the largest splicing differences observed between definitive endoderm and cardiac mesoderm. Integrative multiomics analyses predict lineage-specific RNA binding protein regulators, including a prominent role for Quaking (QKI) in the specification of cardiac mesoderm. Remarkably, knockout of QKI in hESCs disrupts the cardiac mesoderm-associated alternative splicing program and formation of myocytes, likely in part through reduced expression of BIN1 splice variants linked to cardiac development. Our results thus uncover alternative splicing programs associated with the three germ lineages and highlight an important role for QKI and its target transcripts in the formation of cardiac mesoderm.

The Oncogenic Kinase Bcr-Abl Directly Regulates Splicing of BclX through a Quaternary Complex Coordinated by Nck-Beta and Sam-68 Adapter Proteins.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2171-2171
Author(s):  
Bracco Enrico ◽  
Valentina Rosso ◽  
Mussino Stefano ◽  
Francesca Arruga ◽  
Sonia Carturan ◽  
...  
Keyword(s):  
Cell Death ◽  
Alternative Splicing ◽  
Tyrosine Kinase ◽  
Cellular Proliferation ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Alternatively Spliced ◽  
Quaternary Complex

Abstract Abstract 2171 Poster Board II-148 Chronic Myelogenous Leukemia is the prototype of myeloproliferative disorder characterized by a reciprocal chromosomal translocation, involving the chromosomes 9 and 22 —t(9;22)-. The molecular consequence of this translocation is the generation of the Bcr-Abl oncogene that encodes the chimeric Bcr-Abl protein with constitutive tyrosine kinase activity. Its expression in hematopoietic cells induces uncontrolled and growth factor independent cellular proliferation, alteration in cell-cell and cell-matrix adhesion, resistance to apoptosis which altogether are leukemogenesis landmarks. Although it is well established that Bcr-Abl-expressing leukemic cells are highly resistant to apoptotic cell death induced by chemotherapeutic drugs and a number of signaling molecules have been shown to be activated by the Bcr-Abl kinase, the antiapoptotic pathway/s triggered by this oncogene has not been fully understood. The numerous experimental evidences collected in the last years highlight the crucial role played by alternative splicing in the control of apoptosis. Several pre-mRNAs for cell death factors are alternatively spliced, yielding isoforms with opposing functions during programmed cell death. A clear example is Bcl-x transcript which is alternatively spliced to produce the antiapoptotic Bcl-x(L) or the proapoptotic Bcl-x(S). Identical features are shared by another member of the Bcl-2 family, the myeloid cell leukemia-1 (MCL-1) gene which encodes two alternative splicing variants MCL-1S and MCL-1L displaying pro- and anti-apoptotic effects, respectively. CML-derived cell lines overexpress the antiapoptotic protein Bcl-x(L) and their erythroid differentiation is inhibited by Bcl-x(L). The data so far collected indicate that there is an extensive cross-talk among BCL-2 family members by virtue of their protein-protein interactions and the ratio of pro-apoptotic to anti-apoptotic proteins has been shown to be a major detereminant of the cell propensity to undergo apoptosis. Furthermore, it is well established that accelerated and blastic phases of the disease are characterized by deregulated WT1 expression. WT1/KTS- gene encodes for a transcription factor but the WT1/KTS+ isoform has been reported to localize into nuclear speckles, the major sub-nuclear structures enriched pre-messenger RNA and splicing factors. Based on the above premise we started investigating the possibility of an active involvement of Bcr-Abl as candidate regulator of splicing events affecting Bcl-x pre-mRNA. By means of an interactomic approach, based on proteomic strategy using GST-Pull Down assay with an array of SH2 containing proteins, we attempted to gain insight into the role played by adapter molecules and Bcr-Abl in splicing assembling machinery. The data presented aims to demonstrate the presence of quaternary complex involving the SH2-SH3 containing adapter protein Nck-beta, the oncogenic tyrosine kinase Bcr-Abl, the RNA binding protein Sam68, the spliceosome ribonucleprotein hnRNPA1 and WT1. The experimental evidences we have collected support the hypothesis of an Imatinib-dependent interaction occurring between Nck-beta and Bcr-Abl. Furthermore, Pull Down experiments indicate an intermolecular interaction between Nck-beta, Sam68, and hnRNPA1 supporting the idea of a novel complex Bcr-Abl/Nck-beta/Sam68/hnRNPA1/WT1. Biochemical analysis carried-out by Pull-Down experiments has been further corroborated by immunofluorescence staining. RNA Pull Down assay suggest that the quaternary complex Nck-beta/Sam68/hnRNPA1/Bcr-Abl/WT1 might modulates splicing process of Bcl-x gene, whose function has been recently described as crucial in myeloproliferative disorders. Astoningshly, the data collected so far indicates that other mRNAs are pulled-down together with the quaternary complex. Taken together these results represent the first experimental evidences showing an interaction between the oncogene Bcr-Abl and Sam-68 leading to speculate a novel putative role played by Bcr-Abl in the intriguing and complex pre-mRNA splicing scenario. Disclosures: No relevant conflicts of interest to declare.

A saga of cancer epigenetics: linking epigenetics to alternative splicing

2017 ◽  
Vol 474 (6) ◽  
pp. 885-896 ◽  
Author(s):  
Sathiya Pandi Narayanan ◽  
Smriti Singh ◽  
Sanjeev Shukla
Keyword(s):  
Alternative Splicing ◽  
Rna Polymerase Ii ◽  
Cancer Progression ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Epigenetic Modifications ◽  
Protein Isoforms ◽  
Epigenetic Alterations ◽  
Cancer Epigenetics ◽  
Alternatively Spliced

The discovery of an increasing number of alternative splicing events in the human genome highlighted that ∼94% of genes generate alternatively spliced transcripts that may produce different protein isoforms with diverse functions. It is now well known that several diseases are a direct and indirect consequence of aberrant splicing events in humans. In addition to the conventional mode of alternative splicing regulation by ‘cis’ RNA-binding sites and ‘trans’ RNA-binding proteins, recent literature provides enormous evidence for epigenetic regulation of alternative splicing. The epigenetic modifications may regulate alternative splicing by either influencing the transcription elongation rate of RNA polymerase II or by recruiting a specific splicing regulator via different chromatin adaptors. The epigenetic alterations and aberrant alternative splicing are known to be associated with various diseases individually, but this review discusses/highlights the latest literature on the role of epigenetic alterations in the regulation of alternative splicing and thereby cancer progression. This review also points out the need for further studies to understand the interplay between epigenetic modifications and aberrant alternative splicing in cancer progression.

scholarly journals Oncogenic Alternative Splicing Switches: Role in Cancer Progression and Prospects for Therapy

2013 ◽  
Vol 2013 ◽  
pp. 1-17 ◽  
Author(s):  
Serena Bonomi ◽  
Stefania Gallo ◽  
Morena Catillo ◽  
Daniela Pignataro ◽  
Giuseppe Biamonti ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cancer Progression ◽  
Cancer Treatments ◽  
Splicing Regulators ◽  
Transcriptional Regulatory Mechanism ◽  
Transcriptional Regulatory ◽  
Anti Cancer ◽  
Alternatively Spliced ◽  
Splicing Isoforms

Alterations in the abundance or activities of alternative splicing regulators generate alternatively spliced variants that contribute to multiple aspects of tumor establishment, progression and resistance to therapeutic treatments. Notably, many cancer-associated genes are regulated through alternative splicing suggesting a significant role of this post-transcriptional regulatory mechanism in the production of oncogenes and tumor suppressors. Thus, the study of alternative splicing in cancer might provide a better understanding of the malignant transformation and identify novel pathways that are uniquely relevant to tumorigenesis. Understanding the molecular underpinnings of cancer-associated alternative splicing isoforms will not only help to explain many fundamental hallmarks of cancer, but will also offer unprecedented opportunities to improve the efficacy of anti-cancer treatments.

scholarly journals Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal

2015 ◽  
Vol 112 (50) ◽  
pp. 15444-15449 ◽  
Author(s):  
Frida Holm ◽  
Eva Hellqvist ◽  
Cayla N. Mason ◽  
Shawn A. Ali ◽  
Nathaniel Delos-Santos ◽  
...  
Keyword(s):  
Stem Cell ◽  
Alternative Splicing ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Rna Binding ◽  
Combined Treatment ◽  
Embryonic Stem ◽  
Chronic Phase ◽  
Dependent Manner ◽  
Splice Isoform

Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8–10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation–related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination.

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