scholarly journals Cell-specific exon methylation and CTCF binding in neurons regulate calcium ion channel splicing and function

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Eduardo Javier López Soto ◽  
Diane Lipscombe
Keyword(s):  
Alternative Splicing ◽  
Rna Splicing ◽  
Molecular Mechanisms ◽  
Ctcf Binding ◽  
Dna Hypomethylation ◽  
Cell Functions ◽  
Specific Alternative ◽  
Opioid Sensitivity ◽  
And Function

Cell-specific alternative splicing modulates myriad cell functions and is disrupted in disease. The mechanisms governing alternative splicing are known for relatively few genes and typically focus on RNA splicing factors. In sensory neurons, cell-specific alternative splicing of the presynaptic CaV channel Cacna1b gene modulates opioid sensitivity. How this splicing is regulated is unknown. We find that cell and exon-specific DNA hypomethylation permits CTCF binding, the master regulator of mammalian chromatin structure, which, in turn, controls splicing in a DRG-derived cell line. In vivo, hypomethylation of an alternative exon specifically in nociceptors, likely permits CTCF binding and expression of CaV2.2 channel isoforms with increased opioid sensitivity in mice. Following nerve injury, exon methylation is increased, and splicing is disrupted. Our studies define the molecular mechanisms of cell-specific alternative splicing of a functionally validated exon in normal and disease states – and reveal a potential target for the treatment of chronic pain.

scholarly journals Translational suppression via IFG-1/eIF4G confers resistance to stress-induced RNA alternative splicing in Caenorhabditis elegans

2021 ◽  
Author(s):  
Samantha C Chomyshen ◽  
Cheng-Wei Wu
Keyword(s):  
Alternative Splicing ◽  
Caenorhabditis Elegans ◽  
Rna Splicing ◽  
Molecular Mechanisms ◽  
Protein Translation ◽  
Initiation Factor ◽  
A Genome ◽  
Dividing Cells ◽  
Extended Lifespan

Splicing of pre-mRNA is an essential process for dividing cells and splicing defects have been linked to aging and various chronic diseases. Environmental stress has recently been shown to alter splicing fidelity and molecular mechanisms that protect against splicing disruption remains unclear. Using an in vivo RNA splicing reporter, we performed a genome-wide RNAi screen in Caenorhabditis elegans and found that protein translation suppression via silencing of the conserved initiation factor 4G (IFG-1/eIF4G) protects against cadmium-induced splicing disruption. Transcriptome analysis of an ifg-1 deficient mutant revealed an overall increase in splicing fidelity and resistance towards cadmium-induced alternative splicing compared to the wild-type. We found that the ifg-1 mutant up-regulates >80 RNA splicing regulatory genes that are controlled by the TGF-β transcription factor SMA-2. The extended lifespan of the ifg-1 mutant is partially reduced upon sma-2 depletion and completely nullified when core spliceosome genes including snr-1, snr-2, and uaf-2 are knocked down. Together, these data describe a molecular mechanism that provides resistance towards stress-induced alternative splicing and demonstrate an essential role for RNA homeostasis in promoting longevity in a translation-compromised mutant.

scholarly journals Cell-specific exon methylation and CTCF binding in neurons regulates calcium ion channel splicing and function

2019 ◽  
Author(s):  
Eduardo Javier Lopez Soto ◽  
Diane Lipscombe
Keyword(s):  
Alternative Splicing ◽  
Peripheral Nerve ◽  
Calcium Channel ◽  
Nerve Injury ◽  
Calcium Ion ◽  
Peripheral Nerve Injury ◽  
Ctcf Binding ◽  
Channel Gene ◽  
Specific Alternative ◽  
Opioid Sensitivity

SummaryCell-specific alternative splicing modulates myriad cell functions and this process is disrupted in disease. The mechanisms governing alternative splicing are known for relatively few genes and typically focus on RNA splicing factors. In sensory neurons, cell-specific alternative splicing of the presynaptic voltage-gated calcium channel Cacna1b gene modulates opioid sensitivity. How this splicing is regulated has remained unknown. We find that cell-specific exon DNA hypomethylation permits binding of CTCF, the master regulator of chromatin structure in mammals, which, in turn, controls splicing in noxious heat-sensing nociceptors.Hypomethylation of an alternative exon specifically in nociceptors allows for CTCF binding, and expression of CaV2.2 channels with increased opioid sensitivity. Following nerve injury, exon methylation is increased, and splicing is disrupted. Our studies define the molecular mechanisms of cell-specific alternative splicing of a functionally validated exon in normal and disease states – and reveal a potential target for the treatment of chronic pain.HighlightsThe molecular basis of cell-specific splicing of a synaptic calcium channel gene.Splicing controlled by cell-specific exon hypomethylation and CTCF binding.Peripheral nerve injury disrupts exon hypomethylation and splicing.Targeted demethylation of exon by dCAS9-TET modifies alternative splicing.GRAPHICAL ABSTRACTCell-specific epigenetic modifications in a synaptic calcium ion channel gene controls cell-specific splicing in normal and neuropathic pain.In naïve animals, in most neurons, Cacna1b e37a locus is hipermethylated (5-mC) and CTCF does not bind this locus. During splicing, e37a is skipped and Cacna1b mRNAs include e37b. In contrast, in Trpv1-lineage neurons, Cacna1b e37a locus is hypomethylated and is permissive for CTCF binding. CTCF promotes e37a inclusion and both Cacna1b e37a and e37b mRNAs are expressed. E37a confers strong sensitivity to the Cav2.2 channel to inhibition by μ-opioid receptors (μOR). Morphine is more effective at inhibiting e37a-containing Cav2.2 channels. After peripheral nerve injury that results in pathological pain, methylation level of Cacna1b e37a locus is increased, CTCF binding is impaired, and Cacna1b e37a mRNA levels are decreased. This disrupted splicing pattern is associated with reduced efficacy of morphine in vivo.

scholarly journals Multifaceted Functions of Protein Kinase D in Pathological Processes and Human Diseases

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 483
Author(s):  
Xuejing Zhang ◽  
Jaclyn Connelly ◽  
Yapeng Chao ◽  
Qiming Jane Wang
Keyword(s):  
Protein Kinase ◽  
Molecular Mechanisms ◽  
Inflammatory Diseases ◽  
Biological Significance ◽  
Strong Support ◽  
Human Diseases ◽  
Protein Kinase D ◽  
Cell Functions ◽  
And Function

Protein kinase D (PKD) is a family of serine/threonine protein kinases operating in the signaling network of the second messenger diacylglycerol. The three family members, PKD1, PKD2, and PKD3, are activated by a variety of extracellular stimuli and transduce cell signals affecting many aspects of basic cell functions including secretion, migration, proliferation, survival, angiogenesis, and immune response. Dysregulation of PKD in expression and activity has been detected in many human diseases. Further loss- or gain-of-function studies at cellular levels and in animal models provide strong support for crucial roles of PKD in many pathological conditions, including cancer, metabolic disorders, cardiac diseases, central nervous system disorders, inflammatory diseases, and immune dysregulation. Complexity in enzymatic regulation and function is evident as PKD isoforms may act differently in different biological systems and disease models, and understanding the molecular mechanisms underlying these differences and their biological significance in vivo is essential for the development of safer and more effective PKD-targeted therapies. In this review, to provide a global understanding of PKD function, we present an overview of the PKD family in several major human diseases with more focus on cancer-associated biological processes.

scholarly journals The Ubiquitously Expressed Csk Adaptor Protein Cbp Is Dispensable for Embryogenesis and T-Cell Development and Function

2005 ◽  
Vol 25 (23) ◽  
pp. 10533-10542 ◽  
Author(s):  
Marc-Werner Dobenecker ◽  
Christian Schmedt ◽  
Masato Okada ◽  
Alexander Tarakhovsky
Keyword(s):  
T Cell ◽  
Adaptor Protein ◽  
Regulatory Function ◽  
Loss Of Function ◽  
Thymic Development ◽  
Cell Functions ◽  
Carboxy Terminal ◽  
And Function

ABSTRACT Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of “lipid rafts” is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.

Abstract 12192: MBNL1 Directly Regulates the Alternative Splicing of mRNAs That Promote Myofibroblast Differentiation

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Jennifer Davis ◽  
Michelle Sargent ◽  
Jianjian Shi ◽  
Lei Wei ◽  
Maurice S Swanson ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Transforming Growth Factor ◽  
Ventricular Remodeling ◽  
Cardiac Injury ◽  
Myofibroblast Differentiation ◽  
Genome Wide ◽  
A Genome

Rationale: During the cardiac injury response fibroblasts differentiate into myofibroblasts, a cell type that enhances extracellular matrix production and facilitates ventricular remodeling. To better understand the molecular mechanisms whereby myofibroblasts are generated in the heart we performed a genome-wide screen with 18,000 cDNAs, which identified the RNA-binding protein muscleblind-like splicing regulator 1 (MBNL1), suggesting a novel association between mRNA alternative splicing and the regulation of myofibroblast differentiation. Objective: To determine the mechanism whereby MBNL1 regulates myofibroblast differentiation and the cardiac fibrotic response. Methods and Results: Confirming the results from our genome wide screen, adenoviral-mediated overexpression of MBNL1 promoted transformation of rat cardiac fibroblasts and mouse embryonic fibroblasts (MEFs) into myofibroblasts, similar to the level of conversion obtained by the profibrotic agonist transforming growth factor β (TGFβ). Antithetically, Mbnl1 -/- MEFs were refractory to TGFβ-induced myofibroblast differentiation. MBNL1 expression is induced in transforming fibroblasts in response to TGFβ and angiotensin II. These results were extended in vivo by analysis of dermal wound healing, a process dependent on myofibroblast differentiation and their proper activity. By day 6 control mice had achieved 82% skin wound closure compared with only 40% in Mbnl1 -/- mice. Moreover, Mbnl1 -/- mice had reduced survival following myocardial infarction injury due to defective fibrotic scar formation and healing. High throughput RNA sequencing (RNAseq) and RNA immunoprecipitation revealed that MBNL1 directly regulates the alternative splicing of transcripts for myofibroblast signaling factors and cytoskeletal-assembly elements. Functional analysis of these factors as mediators of MBNL1 activity is also described here. Conclusions: Collectively, our data suggest that MBNL1 coordinates myofibroblast transformation by directly mediating the alternative splicing of an array of mRNAs encoding differentiation-specific signaling transcripts, which then alter the fibroblast proteome for myofibroblast structure and function.

scholarly journals DoChaP: The Domain Change Presenter

2020 ◽  
Author(s):  
Shani T. Gal-Oz ◽  
Nimrod Haiat ◽  
Dana Eliyahu ◽  
Guy Shani ◽  
Tal Shay
Keyword(s):  
Alternative Splicing ◽  
Rna Splicing ◽  
Protein Domains ◽  
Visual Presentation ◽  
Structural Effect ◽  
Protein Isoforms ◽  
Multiple Transcripts ◽  
Genome Wide ◽  
Health And Disease ◽  
Specific Alternative

AbstractAlternative RNA splicing results in multiple transcripts of the same gene, possibly encoding for different protein isoforms with different protein domains and functionalities. Whereas it is possible to manually determine the effect of a specific alternative splicing event on the domain composition of a particular encoded protein, the process requires the tedious integration of several data sources; it is therefore error prone and its implementation is not feasible for genome-wide characterization of domains affected by differential splicing. To fulfill the need for an automated solution, we developed the Domain Change Presenter (DoChaP), a web server for the visualization of the exon–domain association. DoChaP visualizes all transcripts of a given gene, the domains of the proteins that they encode, and the exons encoding each domain. The visualization enables a comparison between the transcripts and between the protein isoforms they encode for. The organization and visual presentation of the information makes the structural effect of each alternative splicing event on the protein structure easily identified. To enable a study of the conservation of the exon structure, alternative splicing, and the effect of alternative splicing on protein domains, DoChaP also facilitates an inter-species comparison of domain–exon associations. DoChaP thus provides a unique and easy-to-use visualization of the exon–domain association and its conservation between transcripts and orthologous genes and will facilitate the study of the functional effects of alternative splicing in health and disease.

scholarly journals Molecular mechanisms of olfactory detection in insects: beyond receptors

Open Biology ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 200252
Author(s):  
Hayden R. Schmidt ◽  
Richard Benton
Keyword(s):  
Molecular Mechanisms ◽  
Olfactory Receptors ◽  
Environmental Chemicals ◽  
Ecological Niches ◽  
Cellular Mechanisms ◽  
Signalling Molecules ◽  
Olfactory Systems ◽  
Lymph Fluid ◽  
And Function

Insects thrive in diverse ecological niches in large part because of their highly sophisticated olfactory systems. Over the last two decades, a major focus in the study of insect olfaction has been on the role of olfactory receptors in mediating neuronal responses to environmental chemicals. In vivo , these receptors operate in specialized structures, called sensilla, which comprise neurons and non-neuronal support cells, extracellular lymph fluid and a precisely shaped cuticle. While sensilla are inherent to odour sensing in insects, we are only just beginning to understand their construction and function. Here, we review recent work that illuminates how odour-evoked neuronal activity is impacted by sensillar morphology, lymph fluid biochemistry, accessory signalling molecules in neurons and the physiological crosstalk between sensillar cells. These advances reveal multi-layered molecular and cellular mechanisms that determine the selectivity, sensitivity and dynamic modulation of odour-evoked responses in insects.

scholarly journals HIF-1–dependent repression of equilibrative nucleoside transporter (ENT) in hypoxia

2005 ◽  
Vol 202 (11) ◽  
pp. 1493-1505 ◽  
Author(s):  
Holger K. Eltzschig ◽  
Parween Abdulla ◽  
Edgar Hoffman ◽  
Kathryn E. Hamilton ◽  
Dionne Daniels ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Molecular Mechanisms ◽  
Hypoxia Inducible Factor ◽  
Nucleoside Transporter ◽  
In Vivo Models ◽  
Equilibrative Nucleoside Transporter ◽  
Hypoxia Inducible Factor 1 ◽  
And Function

Extracellular adenosine (Ado) has been implicated as central signaling molecule during conditions of limited oxygen availability (hypoxia), regulating physiologic outcomes as diverse as vascular leak, leukocyte activation, and accumulation. Presently, the molecular mechanisms that elevate extracellular Ado during hypoxia are unclear. In the present study, we pursued the hypothesis that diminished uptake of Ado effectively enhances extracellular Ado signaling. Initial studies indicated that the half-life of Ado was increased by as much as fivefold after exposure of endothelia to hypoxia. Examination of expressional levels of the equilibrative nucleoside transporter (ENT)1 and ENT2 revealed a transcriptionally dependent decrease in mRNA, protein, and function in endothelia and epithelia. Examination of the ENT1 promoter identified a hypoxia inducible factor 1 (HIF-1)–dependent repression of ENT1 during hypoxia. Using in vitro and in vivo models of Ado signaling, we revealed that decreased Ado uptake promotes vascular barrier and dampens neutrophil tissue accumulation during hypoxia. Moreover, epithelial Hif1α mutant animals displayed increased epithelial ENT1 expression. Together, these results identify transcriptional repression of ENT as an innate mechanism to elevate extracellular Ado during hypoxia.

The role of mitochondria in axonal degeneration and tissue repair in MS

2012 ◽  
Vol 18 (8) ◽  
pp. 1058-1067 ◽  
Author(s):  
J van Horssen ◽  
ME Witte ◽  
O Ciccarelli
Keyword(s):  
Mitochondrial Dysfunction ◽  
Tissue Repair ◽  
Molecular Mechanisms ◽  
Axonal Degeneration ◽  
Axonal Damage ◽  
Mitochondrial Metabolism ◽  
Imaging Studies ◽  
And Function

Axonal injury is a key feature of multiple sclerosis (MS) pathology and is currently seen as the main correlate for permanent clinical disability. Although little is known about the pathogenetic mechanisms that drive axonal damage and loss, there is accumulating evidence highlighting the central role of mitochondrial dysfunction in axonal degeneration and associated neurodegeneration. The aim of this topical review is to provide a concise overview on the involvement of mitochondrial dysfunction in axonal damage and destruction in MS. Hereto, we will discuss putative pathological mechanisms leading to mitochondrial dysfunction and recent imaging studies performed in vivo in patients with MS. Moreover, we will focus on molecular mechanisms and novel imaging studies that address the role of mitochondrial metabolism in tissue repair. Finally, we will briefly review therapeutic strategies aimed at improving mitochondrial metabolism and function under neuroinflammatory conditions.

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