scholarly journals QKI is a critical pre-mRNA alternative splicing regulator of cardiac myofibrillogenesis and contractile function

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xinyun Chen ◽  
Ying Liu ◽  
Chen Xu ◽  
Lina Ba ◽  
Zhuo Liu ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
Contractile Function ◽  
Embryonic Stem ◽  
Physiological Role ◽  
Cardiomyocyte Differentiation ◽  
Differentiation Potential ◽  
Splicing Regulator ◽  
Biochemical Analyses ◽  
Normal Differentiation

AbstractThe RNA-binding protein QKI belongs to the hnRNP K-homology domain protein family, a well-known regulator of pre-mRNA alternative splicing and is associated with several neurodevelopmental disorders. Qki is found highly expressed in developing and adult hearts. By employing the human embryonic stem cell (hESC) to cardiomyocyte differentiation system and generating QKI-deficient hESCs (hESCs-QKIdel) using CRISPR/Cas9 gene editing technology, we analyze the physiological role of QKI in cardiomyocyte differentiation, maturation, and contractile function. hESCs-QKIdel largely maintain normal pluripotency and normal differentiation potential for the generation of early cardiogenic progenitors, but they fail to transition into functional cardiomyocytes. In this work, by using a series of transcriptomic, cell and biochemical analyses, and the Qki-deficient mouse model, we demonstrate that QKI is indispensable to cardiac sarcomerogenesis and cardiac function through its regulation of alternative splicing in genes involved in Z-disc formation and contractile physiology, suggesting that QKI is associated with the pathogenesis of certain forms of cardiomyopathies.

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 PSI controls tim splicing and circadian period in Drosophila

2018 ◽  
Author(s):  
Lauren Foley ◽  
Jinli Ling ◽  
Radhika Joshi ◽  
Naveh Evantal ◽  
Sebastian Kadener ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Circadian Rhythms ◽  
Translational Regulation ◽  
Transcriptional Control ◽  
Rna Binding ◽  
P Element ◽  
Peripheral Tissues ◽  
Associated Proteins ◽  
Splicing Regulator ◽  
Post Transcriptional Regulation

AbstractThe Drosophila circadian pacemaker consists of transcriptional feedback loops subjected to both post-transcriptional and post-translational regulation. While post-translational regulatory mechanisms have been studied in detail, much less is known about circadian post-transcriptional control. To have a better understanding of the role and mechanisms of circadian post-transcriptional regulation, we targeted 364 RNA binding and RNA associated proteins with RNA interference. Among the 43 genes we identified was the alternative splicing regulator P-element somatic inhibitor (PSI). PSI downregulation shortens the period of circadian rhythms both in the brain and in peripheral tissues. Interestingly, we found that PSI regulates the thermosensitive alternative splicing of timeless (tim), promoting splicing events favored at warm temperature over those increased at cold temperature. Moreover, the period of circadian behavior was insensitive to PSI downregulation when flies could produce functional TIM proteins only from a transgene that cannot form the thermosensitive splicing isoforms. Therefore, we conclude that PSI regulates the period of Drosophila circadian rhythms through its modulation of the tim splicing pattern.

scholarly journals Phase separation of RNA-binding protein promotes polymerase engagement and transcription

2021 ◽  
Author(s):  
Wen Shao ◽  
Xianju Bi ◽  
Boyang Gao ◽  
Jun Wu ◽  
Yixuan Pan ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Embryonic Stem ◽  
Transcription Control ◽  
Pol Ii ◽  
Genome Wide ◽  
Condensate Formation ◽  
Active Transcription ◽  
Biochemical Analyses

An RNA-involved phase-separation model has been proposed for transcription control. Yet, the molecular links that connect RNA binding to the transcription machinery remain missing. Here we find RNA-binding proteins (RBPs) constitute half of the chromatin proteome in embryonic stem cells (ESCs), and some are colocalized with RNA polymerase (Pol) II at promoters and enhancers. Biochemical analyses of representative RBPs--such as PSPC1 and PTBP1--show that the paraspeckle protein PSPC1 not only prevents the RNA-induced premature release of Pol II, and also makes use of RNA as multivalent molecules to promote Pol II engagement and activity, by enhancing the phase separation and subsequent phosphorylation and release of polymerase condensates. In ESCs, auxin-induced acute degradation of PSPC1 leads to genome-wide defects in Pol II phosphorylation and chromatin-binding and nascent transcription. We propose that the synergistic interplay of RBPs and RNA aids in the rate-limiting step of polymerase condensate formation to promote active transcription.

scholarly journals Hypoxia-induced TGF-β–RBFOX2–ESRP1 axis regulates human MENA alternative splicing and promotes EMT in breast cancer

NAR Cancer ◽  
2020 ◽  
Vol 2 (3) ◽  
Author(s):  
Neha Ahuja ◽  
Cheemala Ashok ◽  
Subhashis Natua ◽  
Deepak Pant ◽  
Anna Cherian ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Alternative Splicing ◽  
Transcriptional Repression ◽  
Rna Binding ◽  
Epithelial Mesenchymal Transition ◽  
Global Changes ◽  
Invasion And Metastasis ◽  
New Paradigm ◽  
Mesenchymal Transition ◽  
Splicing Regulator

Abstract Hypoxic microenvironment heralds epithelial–mesenchymal transition (EMT), invasion and metastasis in solid tumors. Deregulation of alternative splicing (AS) of several cancer-associated genes has been instrumental in hypoxia-induced EMT. Our study in breast cancer unveils a previously unreported mechanism underlying hypoxia-mediated AS of hMENA, a crucial cytoskeleton remodeler during EMT. We report that the hypoxia-driven depletion of splicing regulator ESRP1 leads to skipping of hMENA exon 11a producing a pro-metastatic isoform, hMENAΔ11a. The transcriptional repression of ESRP1 is mediated by SLUG, which gets stimulated via hypoxia-driven TGF-β signaling. Interestingly, RBFOX2, an otherwise RNA-binding protein, is also found to transcriptionally repress ESRP1 while interacting with SLUG. Similar to SLUG, RBFOX2 gets upregulated under hypoxia via TGF-β signaling. Notably, we found that the exosomal delivery of TGF-β contributes to the elevation of TGF-β signaling under hypoxia. Moreover, our results show that in addition to hMENA, hypoxia-induced TGF-β signaling contributes to global changes in AS of genes associated with EMT. Overall, our findings reveal a new paradigm of hypoxia-driven AS regulation of hMENA and insinuate important implications in therapeutics targeting EMT.

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.

scholarly journals PS1 - 158 The Role of LIN28A in Neoplastic Transformation of Human Embryonic Stem Cells (hESCs)

2016 ◽  
Vol 43 (S4) ◽  
pp. S10-S10
Author(s):  
R. Kaur ◽  
L. Liang ◽  
T. Werbowetski-Ogilvie
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Rna Binding ◽  
Embryonic Stem ◽  
Neoplastic Transformation ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Human Cancers

Human embryonic stem cells (hESCs) are known for their indefinite self-renewal ability and pluripotent nature. However, during long-term culture, normal hESCs can undergo neoplastic transformation and acquire enhanced self-renewal ability and aberrant differentiation potential. These transformed-hESCs (trans-hESCs) exhibit high expression of the pluripotent gene, LIN28A. LIN28A, an RNA binding protein, is known: for its role in self-renewal of hESCs, as a reprogramming factor for generating induced-pluripotent stem cells and as a potent oncogene in several poorly differentiated, highly malignant human cancers. Despite its multiple functions, how LIN28A contributes to neoplastic transformation of normal hESCs is poorly understood. Our preliminary data demonstrate that following LIN28A knockdown, trans-hESCs display normal hESCs morphology consisting of both pluripotent colony cells surrounded by more differentiated fibroblast-like cells. Neural precursors derived from LIN28A knockdown trans-hESCs also revert back to a state of normal cell morphology and growth. Further analyses revealed that the expression levels of stage-specific embryonic antigen (SSEA3), OCT3/4 and NANOG decreases and are comparable to that observed in normal hESCs following LIN28A downregulation. Expression of miRNA targets of LIN28A such as let7i and mir125b was increased to levels seen in normal hESCs. These preliminary results indicate that LIN28A is a major contributing factor to neoplastic transformation of hESCs and that this process can be reversed by cellular “reprogramming”. This study will enhance our understanding of role of LIN28A in the transformation process in various human cancers thus, underscoring the value of hESCs and their neoplastic-derivatives as cellular and molecular model for studying tumor progression.

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.

scholarly journals The RNA-Binding Protein SAM68 regulates cardiomyocyte differentiation by enhancing Gata4 translation

2022 ◽  
Author(s):  
Alessandro Dasti ◽  
Maria Carla Antonelli ◽  
Magdalena Arnal Segura ◽  
Alexandros Armaos ◽  
Sarah Bonnin ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Embryonic Stem ◽  
Ribosome Profiling ◽  
Cardiomyocyte Differentiation ◽  
Mammalian Development ◽  
Mobility Shift ◽  
Lineage Specification ◽  
Rna Immunoprecipitation ◽  
Mobility Shift Assay

The signal transduction and activation of RNA (STAR) family is composed of RNA-binding proteins (RBPs) that play a central role in mammalian development. Nonetheless, the functions and modes of action that STAR proteins have in lineage specification are still poorly understood. Here, we characterized the role of STAR proteins SAM68 and QUAKING (QKI) in pluripotency and differentiation by performing their depletion through CRISPR-Cas9 in mouse embryonic stem cells (mESCs). Combining RNA-sequencing, ribosome profiling and advanced computational predictions, we found that both SAM68 and QKI regulate the mESCs self-renewal and are indispensable for cardiomyocyte differentiation. At the molecular level, we discovered that SAM68 and QKI antagonistically control the expression of cardiogenic factors. Our calculations indicated that SAM68, unlike QKI, binds the cardiogenic-specific transcription factor Gata4 in a region spanning nucleotides 500 to 1000 of the mRNA corresponding to part of the 5' untranslated region and the first exon. We validated the predictions by electrophoretic mobility shift assay and RNA immunoprecipitation showing that SAM68 controls the translation of Gata4 during mESCs differentiation towards the cardiomyocyte lineage.

scholarly journals A tumor-associated splice-isoform ofMAP2K7drives dedifferentiation in MBNL1-low cancers via JNK activation

2020 ◽  
Vol 117 (28) ◽  
pp. 16391-16400 ◽  
Author(s):  
Debleena Ray ◽  
Yu Chye Yun ◽  
Muhammad Idris ◽  
Shanshan Cheng ◽  
Arnoud Boot ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Embryonic Stem ◽  
The Cancer Genome Atlas ◽  
Down Regulation ◽  
Poor Overall Survival ◽  
Splicing Regulator ◽  
Cancer Genome Atlas ◽  
Jnk Activation

Master splicing regulator MBNL1 shapes large transcriptomic changes that drive cellular differentiation during development. Here we demonstrate that MBNL1 is a suppressor of tumor dedifferentiation. We surveyedMBNL1expression in matched tumor/normal pairs across The Cancer Genome Atlas and found thatMBNL1was down-regulated in several common cancers. Down-regulation ofMBNL1predicted poor overall survival in breast, lung, and stomach adenocarcinomas and increased relapse and distant metastasis in triple-negative breast cancer. Down-regulation of MBNL1 led to increased tumorigenic and stem/progenitor-like properties in vitro and in vivo. A discrete set of alternative splicing events (ASEs) are shared betweenMBNL1-low cancers and embryonic stem cells including aMAP2K7∆exon2 splice variant that leads to increased stem/progenitor-like properties via JNK activation. Accordingly, JNK inhibition is capable of reversingMAP2K7∆exon2-driven tumor dedifferentiation in MBNL1-low cancer cells. Our work elucidates an alternative-splicing mechanism that drives tumor dedifferentiation and identifies biomarkers that predict enhanced susceptibility to JNK inhibition.

scholarly journals RNA Binding Protein Ptbp2 Is Essential for Male Germ Cell Development

2015 ◽  
Vol 35 (23) ◽  
pp. 4030-4042 ◽  
Author(s):  
Leah L. Zagore ◽  
Sarah E. Grabinski ◽  
Thomas J. Sweet ◽  
Molly M. Hannigan ◽  
R. Michael Sramkoski ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Germ Cell ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Cell Loss ◽  
Cell Development ◽  
Splicing Regulation ◽  
Germ Cell Development ◽  
Splicing Regulator ◽  
Alternatively Spliced

RNA binding proteins (RBPs) are increasingly recognized as essential factors in tissue development and homeostasis. The polypyrimidine tract binding (PTB) protein family of RBPs are important posttranscriptional regulators of gene expression. In the nervous system, the function and importance of PTB protein 2 (Ptbp2) as a key alternative splicing regulator is well established. Ptbp2 is also abundantly expressed during spermatogenesis, but its role in this developmental program has not been explored. Additionally, the importance of alternative splicing regulation in spermatogenesis is unclear. Here, we demonstrate that Ptbp2 is essential for spermatogenesis. We also describe an improved dual fluorescence flow cytometry strategy to discriminate, quantify, and collect germ cells in different stages of development. Using this approach, in combination with traditional histological methods, we show that Ptbp2 ablation results in germ cell loss due to increased apoptosis of meiotic spermatocytes and postmeiotic arrest of spermatid differentiation. Furthermore, we show that Ptbp2 is required for alternative splicing regulation in the testis, as in brain. Strikingly, not all of the alternatively spliced RNAs examined were sensitive to Ptbp2 loss in both tissues. Collectively, the data provide evidence for an important role for alternative splicing regulation in germ cell development and a central role for Ptbp2 in this process.

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