scholarly journals PCIF1 catalyzes m6Am mRNA methylation to regulate gene expression

2018 ◽  
Author(s):  
Erdem Sendinc ◽  
David Valle-Garcia ◽  
Abhinav Dhall ◽  
Hao Chen ◽  
Telmo Henriques ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Regulation ◽  
List Type ◽  
Methylation Analysis ◽  
Rna Pol Ii ◽  
Regulate Gene Expression ◽  
Evolutionarily Conserved ◽  
Mrna Methylation

SummarymRNA modifications play an important role in regulating gene expression. One of the most abundant mRNA modifications is N6,2-O-dimethyladenosine (m6Am). Here, we demonstrate that m6Am is an evolutionarily conserved mRNA modification mediated by the Phosphorylated CTD Interacting Factor 1 (PCIF1), which catalyzes m6A methylation on 2-O-methylated adenine located at the 5’ ends of mRNAs. Furthermore, PCIF1 catalyzes only 5’ m6Am methylation of capped mRNAs, but not internal m6A methylation in vitro and in vivo. Our global mRNA methylation analysis revealed that there is no crosstalk between m6Am and m6A mRNA methylation events, suggesting that m6Am is functionally distinct from m6A. Importantly, our data indicate that m6Am negatively impacts translation of methylated mRNAs by antagonizing cap binding protein eIF4E. Together, we identify the first and only human mRNA m6Am methyltransferase and demonstrate a novel mechanism of gene expression regulation through PCIF1-mediated m6Am mRNA methylation in eukaryotes.HighlightsPCIF1 is an evolutionarily conserved mRNA m6Am methyltransferaseLoss of PCIF1 leads to a complete loss of m6Am, whereas m6A level and distribution are not affectedPCIF1 mediated m6Am does not affect RNA Pol II transcription or mRNA stabilitym6Am-Exo-Seq is a robust methodology that enables global m6Am mappingm6Am suppresses cap dependent translation

scholarly journals Mediator complex subunit Med19 binds directly GATA transcription factors and is required with Med1 for GATA-driven gene regulation in vivo

2020 ◽  
Vol 295 (39) ◽  
pp. 13617-13629
Author(s):  
Clément Immarigeon ◽  
Sandra Bernat-Fabre ◽  
Emmanuelle Guillou ◽  
Alexis Verger ◽  
Elodie Prince ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Target Genes ◽  
Direct Interaction ◽  
Mediator Complex ◽  
Loss Of Function ◽  
Transcriptional Responses ◽  
Rna Pol Ii ◽  
Evolutionarily Conserved

The evolutionarily conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Pol II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. But are these binary partnerships sufficient to mediate TF functions? We have previously established that the Med1 Mediator subunit serves as a cofactor of GATA TFs in Drosophila, as shown in mammals. Here, we observe mutant phenotype similarities between another subunit, Med19, and the Drosophila GATA TF Pannier (Pnr), suggesting functional interaction. We further show that Med19 physically interacts with the Drosophila GATA TFs, Pnr and Serpent (Srp), in vivo and in vitro through their conserved C-zinc finger domains. Moreover, Med19 loss of function experiments in vivo or in cellulo indicate that it is required for Pnr- and Srp-dependent gene expression, suggesting general GATA cofactor functions. Interestingly, Med19 but not Med1 is critical for the regulation of all tested GATA target genes, implying shared or differential use of MED subunits by GATAs depending on the target gene. Lastly, we show a direct interaction between Med19 and Med1 by GST pulldown experiments indicating privileged contacts between these two subunits of the MED middle module. Together, these findings identify Med19/Med1 as a composite GATA TF interface and suggest that binary MED subunit–TF partnerships are probably oversimplified models. We propose several mechanisms to account for the transcriptional regulation of GATA-targeted genes.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

2021 ◽  
Author(s):  
Yu Takahashi ◽  
Yu Inoue ◽  
Keitaro Kuze ◽  
Shintaro Sato ◽  
Makoto Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Gene Expression Regulation ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

Epigenetherapy, a new concept

2011 ◽  
Vol 2 (3) ◽  
pp. 127-134
Author(s):  
Tiia Husso ◽  
Mikko P. Turunen ◽  
Nigel Parker ◽  
Seppo Ylä-Herttuala
Keyword(s):  
Gene Expression ◽  
Small Rnas ◽  
Basic Research ◽  
Clinical Applications ◽  
Regulate Gene Expression ◽  
Endogenous Gene ◽  
Rna Molecules ◽  
Regulate Gene

AbstractSmall RNAs have been shown to regulate gene transcription by interacting with the promoter region and modifying the histone code. The exact mechanism of function is still unclear but the feasibility to activate or repress endogenous gene expression with small RNA molecules has already been demonstrated in vitro and in vivo. In traditional gene therapy non-mutated or otherwise useful genes are inserted into patient's cells to treat a disease. In epigenetherapy the action of small RNAs is utilized by delivering only the small RNAs to patient's cells where they then regulate gene expression by epigenetic mechanisms. This method could be widely useful not only for basic research but also for clinical applications of small RNAs.

scholarly journals A novel G-quadruplex motif in the Human MET promoter region

2017 ◽  
Vol 37 (6) ◽  
Author(s):  
Jing Yan ◽  
Deming Zhao ◽  
Liping Dong ◽  
Shuang Pan ◽  
Fengjin Hao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Regulation ◽  
Luciferase Assay ◽  
Klenow Fragment ◽  
Regulate Gene Expression ◽  
Physiological Conditions ◽  
Quadruplex Structure ◽  
G Quadruplex ◽  
Quadruplex Formation

It is known that the guanine-rich strands in proto-oncogene promoters can fold into G-quadruplex structures to regulate gene expression. An intramolecular parallel G-quadruplex has been identified in MET promoter. It acts as a repressor in regulating MET expression. However, the full guanine-rich region in MET promoter forms a hybrid parallel/antiparallel G-quadruplex structure under physiological conditions, which means there are some antiparallel and hybrid parallel/antiparallel G-quadruplex structures in this region. In the present study, our data indicate that g3-5 truncation adopts an intramolecular hybrid parallel/antiparallel G-quadruplex under physiological conditions in vitro. The g3-5 G-quadruplex structure significantly stops polymerization by Klenow fragment in K+ buffer. Furthermore, the results of circular dichroism (CD) spectra and polymerase stop assay directly demonstrate that the G-quadruplex structure in g3-5 fragment can be stabilized by the G-quadruplex ligand TMPyP4 (5,10,15,20-tetra-(N-methyl-4-pyridyl) porphine). But the dual luciferase assay indicates TMPyP4 has no effect on the formation of g3-5 G-quadruplex in HepG2 cells. The findings in the present study will enrich our understanding of the G-quadruplex formation in proto-oncogene promoters and the mechanisms of gene expression regulation.

scholarly journals Evolutionarily Conserved Long Non-coding RNA Regulates Gene Expression in Cytokine Storm During COVID-19

2021 ◽  
Vol 8 ◽  
Author(s):  
Olanrewaju B. Morenikeji ◽  
Kahleel Bernard ◽  
Ellis Strutton ◽  
Madeleine Wallace ◽  
Bolaji N. Thomas
Keyword(s):  
Gene Expression ◽  
Drug Targets ◽  
Regulatory Elements ◽  
Cytokine Storm ◽  
Cellular Processes ◽  
Non Coding Rna ◽  
Evolutionarily Conserved ◽  
Multiple Species

Coronavirus is a family of viruses including alpha-, beta-, gamma-, delta-coronaviruses. Only alpha- and betacoronaviruses have been observed to infect humans. Past outbreaks of SARS-CoV and MERS-CoV, both betacoronavirus, are the result of a spillover from animals. Recently, a new strain termed SARS-CoV-2 emerged in December 2019 in Wuhan, China. Severe cases of COVID-19, the disease caused by SARS-CoV-2, lead to acute respiratory distress syndrome (ARDS). One contributor to the development of ARDS is cytokine storm, an overwhelming inflammatory immune response. Long non-coding RNAs (lncRNAs) are genetic regulatory elements that, among many functions, alter gene expression and cellular processes. lncRNAs identified to be pertinent in COVID-19 cytokine storm have the potential to serve as disease markers or drug targets. This project aims to computationally identify conserved lncRNAs potentially regulating gene expression in cytokine storm during COVID-19. We found 22 lncRNAs that can target 10 cytokines overexpressed in COVID-19 cytokine storm, 8 of which targeted two or more cytokine storm cytokines. In particular, the lncRNA non-coding RNA activated by DNA damage (NORAD), targeted five out of the ten identified cytokine storm cytokines, and is evolutionarily conserved across multiple species. These lncRNAs are ideal candidates for further in vitro and in vivo analysis.

scholarly journals The Causes and Consequences of miR-503 Dysregulation and Its Impact on Cardiovascular Disease and Cancer

2021 ◽  
Vol 12 ◽  
Author(s):  
Yanjing He ◽  
Yin Cai ◽  
Pearl Mingchu Pai ◽  
Xinling Ren ◽  
Zhengyuan Xia
Keyword(s):  
Gene Expression ◽  
Cardiovascular Disease ◽  
Diagnostic Marker ◽  
Translational Repression ◽  
Biological Processes ◽  
Regulate Gene Expression ◽  
Non Coding Rnas ◽  
And Oxidative Stress

microRNAs (miRs) are short, non-coding RNAs that regulate gene expression by mRNA degradation or translational repression. Accumulated studies have demonstrated that miRs participate in various biological processes including cell differentiation, proliferation, apoptosis, metabolism and development, and the dysregulation of miRs expression are involved in different human diseases, such as neurological, cardiovascular disease and cancer. microRNA-503 (miR-503), one member of miR-16 family, has been studied widely in cardiovascular disease and cancer. In this review, we summarize and discuss the studies of miR-503 in vitro and in vivo, and how miR-503 regulates gene expression from different aspects of pathological processes of diseases, including carcinogenesis, angiogenesis, tissue fibrosis and oxidative stress; We will also discuss the mechanisms of dysregulation of miR-503, and whether miR-503 could be applied as a diagnostic marker or therapeutic target in cardiovascular disease or cancer.

scholarly journals Optimization of a bacterial three-hybrid assay through in vivo titration of an RNA-DNA adapter-protein

2020 ◽  
Author(s):  
Clara D. Wang ◽  
Rachel Mansky ◽  
Hannah LeBlanc ◽  
Chandra M. Gravel ◽  
Katherine E. Berry
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Regulate Gene Expression ◽  
E Coli ◽  
Binding Energetics ◽  
Response To Stress ◽  
First Time ◽  
Regulate Gene

ABSTRACTNon-coding RNAs regulate gene expression in every domain of life. In bacteria, small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by RNA-chaperone proteins, such as Hfq. We have recently developed a bacterial three-hybrid (B3H) assay that detects the strong binding interactions of certain E. coli sRNAs with proteins Hfq and ProQ. Despite the promise of this system, the signal-to-noise has made it challenging to detect weaker interactions. In this work, we use Hfq-sRNA interactions as a model system to optimize the B3H assay, so that weaker RNA-protein interactions can be more reliably detected. We find that the concentration of the RNA-DNA adapter is an important parameter in determining the signal in the system, and have modified the plasmid expressing this component to tune its concentration to optimal levels. In addition, we have systematically perturbed the binding affinity of Hfq-RNA interactions to define, for the first time, the relationship between B3H signal and in vitro binding energetics. The new pAdapter construct presented here substantially expands the range of detectable interactions in the B3H assay, broadening its utility. This improved assay will increase the likelihood of identifying novel protein-RNA interactions with the B3H system, and will facilitate exploration of the binding mechanisms of these interactions.

scholarly journals In Vivo Behavior of the Tandem Glycine Riboswitch in Bacillus subtilis

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Arianne M. Babina ◽  
Nicholas E. Lea ◽  
Michelle M. Meyer
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Regulate Gene Expression ◽  
Binding Domains ◽  
Expression Platform ◽  
The Impact ◽  
Organismal Fitness ◽  
Regulate Gene

ABSTRACT In many bacterial species, the glycine riboswitch is composed of two homologous ligand-binding domains (aptamers) that each bind glycine and act together to regulate the expression of glycine metabolic and transport genes. While the structure and molecular dynamics of the tandem glycine riboswitch have been the subject of numerous in vitro studies, the in vivo behavior of the riboswitch remains largely uncharacterized. To examine the proposed models of tandem glycine riboswitch function in a biologically relevant context, we characterized the regulatory activity of mutations to the riboswitch structure in Bacillus subtilis using β-galactosidase assays. To assess the impact disruptions to riboswitch function have on cell fitness, we introduced these mutations into the native locus of the tandem glycine riboswitch within the B. subtilis genome. Our results indicate that glycine does not need to bind both aptamers for regulation in vivo and mutations perturbing riboswitch tertiary structure have the most severe effect on riboswitch function and gene expression. We also find that in B. subtilis, the glycine riboswitch-regulated gcvT operon is important for glycine detoxification. IMPORTANCE The glycine riboswitch is a unique cis-acting mRNA element that contains two tandem homologous glycine-binding domains that act on a single expression platform to regulate gene expression in response to glycine. While many in vitro experiments have characterized the tandem architecture of the glycine riboswitch, little work has investigated the behavior of this riboswitch in vivo. In this study, we analyzed the proposed models of tandem glycine riboswitch regulation in the context of its native locus within the Bacillus subtilis genome and examined how disruptions to glycine riboswitch function impact organismal fitness. Our work offers new insights into riboswitch function in vivo and reinforces the potential of riboswitches as novel antimicrobial targets. IMPORTANCE The glycine riboswitch is a unique cis-acting mRNA element that contains two tandem homologous glycine-binding domains that act on a single expression platform to regulate gene expression in response to glycine. While many in vitro experiments have characterized the tandem architecture of the glycine riboswitch, little work has investigated the behavior of this riboswitch in vivo. In this study, we analyzed the proposed models of tandem glycine riboswitch regulation in the context of its native locus within the Bacillus subtilis genome and examined how disruptions to glycine riboswitch function impact organismal fitness. Our work offers new insights into riboswitch function in vivo and reinforces the potential of riboswitches as novel antimicrobial targets.

scholarly journals Entrainment of circadian rhythms depends on firing rates and neuropeptide release of VIP SCN neurons

2018 ◽  
Author(s):  
Cristina Mazuski ◽  
John H. Abel ◽  
Samantha P. Chen ◽  
Tracey O. Hermanstyne ◽  
Jeff R. Jones ◽  
...  
Keyword(s):  
Gene Expression ◽  
Circadian Rhythms ◽  
Time Of Day ◽  
Neuronal Firing ◽  
List Type ◽  
Dependent Manner ◽  
Firing Activity ◽  
Firing Patterns

SummaryThe mammalian suprachiasmatic nucleus (SCN) functions as a master circadian pacemaker, integrating environmental input to align physiological and behavioral rhythms to local time cues. Approximately 10% of SCN neurons express vasoactive intestinal polypeptide (VIP); however, it is unknown how firing activity of VIP neurons releases VIP to entrain circadian rhythms. To identify physiologically relevant firing patterns, we optically tagged VIP neurons and characterized spontaneous firing over three days. VIP neurons had circadian rhythms in firing rate and exhibited two classes of instantaneous firing activity. We next tested whether physiologically relevant firing affected circadian rhythms through VIP release. We found that VIP neuron stimulation with high, but not low, frequencies shifted gene expression rhythms in vitro through VIP signaling. In vivo, high frequency VIP neuron activation rapidly entrained circadian locomotor rhythms. Thus, increases in VIP neuronal firing frequency release VIP and entrain molecular and behavioral circadian rhythms.HighlightsMazuski et al. identified three classes of circadian SCN neurons based on their distinct firing patterns consistent over multiple daysThere are two distinct classes (tonic and irregular firing) of VIP SCN neurons.Stimulation of VIP SCN neurons at physiologically relevant frequencies phase shifts whole-SCN circadian rhythms in gene expression through VIP release. These effects are blocked with VIP antagonists.Firing of VIP SCN neurons entrains circadian rhythms in locomotor behavior in a frequency and time-of-day dependent manner.

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