Analysis of post-transcriptional regulation using the FunREG method

2010 ◽  
Vol 38 (6) ◽  
pp. 1608-1614 ◽  
Author(s):  
Benoît Laloo ◽  
Marion Maurel ◽  
Sandra Jalvy-Delvaille ◽  
Francis Sagliocco ◽  
Christophe F. Grosset
Keyword(s):  
Transcriptional Regulation ◽  
Translation Efficiency ◽  
Histone Deacetylase 6 ◽  
Reporter System ◽  
Rna Sequences ◽  
Experimental Conditions ◽  
Fluorescent Reporter ◽  
Wide Range ◽  
Transcriptional Deregulation ◽  
Post Transcriptional Regulation

An increasing number of arguments, including altered microRNA expression, support the idea that post-transcriptional deregulation participates in gene disturbances found in diseased tissues. To evaluate this hypothesis, we developed a method which facilitates post-transcriptional investigations in a wide range of human cells and experimental conditions. This method, called FunREG (functional, integrated and quantitative method to measure post-transcriptional regulation), connects lentiviral transduction with a fluorescent reporter system and quantitative PCR. Using FunREG, we efficiently measured post-transcriptional regulation mediated either by selected RNA sequences or regulatory factors (microRNAs), and then evaluated the contribution of mRNA decay and translation efficiency in the observed regulation. We demonstrated the existence of gene-specific post-transcriptional deregulation in liver tumour cells, and also reported a molecular link between a transcript variant abrogating HDAC6 (histone deacetylase 6) regulation by miR-433 and a rare familial genetic disease. Because FunREG is sensitive, quantitative and easy to use, many applications can be envisioned in fundamental and pathophysiological research.

scholarly journals Genome-wide dynamics of RNA synthesis, processing and degradation without RNA metabolic labeling

2019 ◽  
Author(s):  
Mattia Furlan ◽  
Eugenia Galeota ◽  
Nunzio Del Gaudio ◽  
Erik Dassi ◽  
Michele Caselle ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Steady State ◽  
Time Course ◽  
Rna Synthesis ◽  
Computational Method ◽  
Metabolic Labeling ◽  
Experimental Conditions ◽  
Rna Dynamics ◽  
Kinetic Rates ◽  
Post Transcriptional Regulation

AbstractThe kinetic rates of RNA synthesis, processing and degradation determine the dynamics of transcriptional regulation by governing both the abundance and the responsiveness to modulations of premature and mature RNA species. The study of RNA dynamics is largely based on the integrative analysis of total and nascent transcription, with the latter being quantified through RNA metabolic labeling. We describe here INSPEcT-, a computational method based on mathematical modeling of intronic and exonic expression, able to derive the dynamics of transcription from steady state or time course profiling of just total RNA, without requiring any information on nascent transcripts. Our approach closely recapitulates the kinetic rates obtained through RNA metabolic labeling, improves the ability to detect changes in transcripts half-lives, reduces the cost and complexity of the experiments, and can be adopted to study experimental conditions where nascent transcription cannot be readily profiled. Finally, we applied INSPEcT- to the characterization of post-transcriptional regulation landscapes in dozens of physiological and disease conditions. This approach was included in the INSPEcT Bioconductor package, which can now unveil RNA dynamics from steady state or time course data, with or without the profiling of nascent RNA.

scholarly journals Small RNA Regulation of Virulence in Pathogenic Escherichia coli

2021 ◽  
Vol 10 ◽  
Author(s):  
Brandon M. Sy ◽  
Jai J. Tree
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Regulation ◽  
Virulence Factors ◽  
Small Rna ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Human Pathogens ◽  
Rna Regulation ◽  
Wide Range ◽  
Post Transcriptional Regulation

Enteric and extraintestinal pathotypes of Escherichia coli utilize a wide range of virulence factors to colonize niches within the human body. During infection, virulence factors such as adhesins, secretions systems, or toxins require precise regulation and coordination to ensure appropriate expression. Additionally, the bacteria navigate rapidly changing environments with fluctuations in pH, temperature, and nutrient levels. Enteric pathogens utilize sophisticated, interleaved systems of transcriptional and post-transcriptional regulation to sense and respond to these changes and modulate virulence gene expression. Regulatory small RNAs and RNA-binding proteins play critical roles in the post-transcriptional regulation of virulence. In this review we discuss how the mosaic genomes of Escherichia coli pathotypes utilize small RNA regulation to adapt to their niche and become successful human pathogens.

scholarly journals Regulation of Fetal Genes by Transitions among RNA-Binding Proteins during Liver Development

2020 ◽  
Vol 21 (23) ◽  
pp. 9319
Author(s):  
Toru Suzuki ◽  
Shungo Adachi ◽  
Chisato Kikuguchi ◽  
Shinsuke Shibata ◽  
Saori Nishijima ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fetal Liver ◽  
Regulatory Protein ◽  
Rna Decay ◽  
Translation Efficiency ◽  
Mrna Binding ◽  
Post Transcriptional Regulation

Transcripts of alpha-fetoprotein (Afp), H19, and insulin-like growth factor 2 (Igf2) genes are highly expressed in mouse fetal liver, but decrease drastically during maturation. While transcriptional regulation of these genes has been well studied, the post-transcriptional regulation of their developmental decrease is poorly understood. Here, we show that shortening of poly(A) tails and subsequent RNA decay are largely responsible for the postnatal decrease of Afp, H19, and Igf2 transcripts in mouse liver. IGF2 mRNA binding protein 1 (IMP1), which regulates stability and translation efficiency of target mRNAs, binds to these fetal liver transcripts. When IMP1 is exogenously expressed in mouse adult liver, fetal liver transcripts show higher expression and possess longer poly(A) tails, suggesting that IMP1 stabilizes them. IMP1 declines concomitantly with fetal liver transcripts as liver matures. Instead, RNA-binding proteins (RBPs) that promote RNA decay, such as cold shock domain containing protein E1 (CSDE1), K-homology domain splicing regulatory protein (KSRP), and CUG-BP1 and ETR3-like factors 1 (CELF1), bind to 3′ regions of fetal liver transcripts. These data suggest that transitions among RBPs associated with fetal liver transcripts shift regulation from stabilization to decay, leading to a postnatal decrease in those fetal transcripts.

scholarly journals Salmonella endorses a dormant state within human epithelial cells for persistent infection

2020 ◽  
Author(s):  
Chak Hon Luk ◽  
Yuen-Yan Chang ◽  
Jost Enninga
Keyword(s):  
Flow Cytometry ◽  
Epithelial Cells ◽  
Fluorescence Microscopy ◽  
Host Cell ◽  
Cell Types ◽  
Host Cells ◽  
Cell Type ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Wide Range

AbstractSalmonella Typhimurium (S. Typhimurium) is an enteric bacterium capable of invading a wide range of hosts, including rodents and humans. It targets different host cell types showing different intracellular lifestyles. Within the infected cells S. Typhimurium colonizes multiple intracellular niches, and it is able to either actively divide at various rates, or remain dormant to persist. A comprehensive tool to monitor these distinct S. Typhimurium lifestyles has not been available so far. Here we developed a novel fluorescent reporter, Salmonella Intracellular Analyzer (SINA), compatible for fluorescence microscopy and flow cytometry for quantification at the single-bacterium level. Using SINA, we identified a S. Typhimurium subpopulation in infected epithelial cells that exhibits a unique phenotype in comparison to the previously documented vacuolar or cytosolic S. Typhimurium. This newly identified subpopulation remained dormant within a vesicular compartment distinct from either conventional Salmonella-containing vacuoles (SCV) or the previously reported niche of dormant S. Typhimurium inside macrophages. The dormant S. Typhimurium inside enterocytes were viable and expressed Salmonella Pathogenicity Island 2 (SPI-2) virulence factors at later infection time points. We found that the formation of these dormant S. Typhimurium is not triggered by the loss of SPI-2 expression but it is regulated by (p)ppGpp-mediated stringent response through RelA and SpoT. We predict that intraepithelial dormant S. Typhimurium represents an important pathogen niche as it provides an alternative strategy for S. Typhimurium pathogenicity and persistence.Author SummarySalmonella Typhimurium is a clinically relevant bacterial pathogen that causes Salmonellosis. It can actively or passively invade various host cell types and reside in a Salmonella-containing vacuole (SCV) within host cells. The SCV can be remodeled into a replicative niche with the aid of Salmonella Type III Secretion System 2 (T3SS2) effectors or else, the SCV is ruptured for the access of the nutrient-rich host cytosol. Depending on the infected host cell type, S. Typhimurium undertake different lifestyles that are distinct by their subcellular localization, replication rate and metabolic rate. We present here a novel fluorescent reporter system that rapidly detects S. Typhimurium lifestyles using fluorescence microscopy and flow cytometry. We identified a dormant S. Typhimurium population within enterocytes that displays capacities in host cell persistence, dormancy exit and antibiotic tolerance. We found that the molecular pathway suppressing S. Typhimurium dormancy in enterocytes is the one that has been shown to promote dormancy in macrophages. This suggests a divergent physiological consequence regulated by the same set of S. Typhimurium molecular mediators depending on the challenged host cell type. Altogether, our work demonstrates the potential of fluorescence reporters in facile bacterial characterization, and revealed a dormant S. Typhimurium population in human enterocytes that is distinct from those observed in macrophages and fibroblasts.

scholarly journals phiC31 integrase for recombination mediated single copy insertion and genome manipulation in C. elegans

Genetics ◽  
2021 ◽  
Author(s):  
Fang-Jung Yang ◽  
Chiao-Nung Chen ◽  
Tiffany Chang ◽  
Ting-Wei Cheng ◽  
Ni-Chen Chang ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Integration Method ◽  
Single Copy ◽  
Large Set ◽  
Reporter System ◽  
Phic31 Integrase ◽  
C Elegans ◽  
Cassette Exchange ◽  
Post Transcriptional Regulation ◽  
Genome Manipulation

Abstract C. elegans benefits from a large set of tools for genome manipulation. Yet, the precise single-copy insertion of very large DNA constructs (>10 kb) and the generation of inversions are still challenging. Here, we adapted the phiC31 integrase system for C. elegans. We generated an integrated phiC31 integrase expressing strain flanked by attP sites that serves as a landing pad for integration of transgenes by recombination mediated cassette exchange (RCME). This strain is unc-119(-) so RMCE integrants can be produced simply by injection of a plasmid carrying attB sites flanking unc-119(+) and the gene(s) of interest. Additionally, phiC31 integrase is removed concomitantly with integration, eliminating the need to outcross away the integrase. Integrations were obtained for insert sizes up to ∼33.4 kb. Taking advantage of this integration method we establish a dual color fluorescent operon reporter system able to study post-transcriptional regulation of mRNA. Last, we show that large chromosomal segments can be inverted using phiC31 integrase. Thus, the phiC31 integrase system should be a useful addition to the C. elegans toolkit.

scholarly journals Dietary restriction induces post-transcriptional regulation of longevity genes

2019 ◽  
Author(s):  
Jarod A. Rollins ◽  
Santina S. Snow ◽  
Pankaj Kapahi ◽  
Aric N. Rogers
Keyword(s):  
Transcriptional Regulation ◽  
Dietary Restriction ◽  
Translational Regulation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Intron Retention ◽  
Translation Efficiency ◽  
Nonsense Mediated Decay ◽  
Longevity Genes ◽  
Post Transcriptional Regulation

AbstractDietary restriction increases lifespan through adaptive changes in gene expression. To understand more about these changes, we analyzed the transcriptome and translatome of C. elegans subjected to dietary restriction. Transcription of muscle regulatory and structural genes increased, while increased expression of amino acid metabolism and neuropeptide signaling genes was controlled at the level of translation. Evaluation of post-transcriptional regulation identified putative roles for RNA binding proteins, RNA editing, microRNA, alternative splicing, and nonsense mediated decay in response to nutrient limitation. Using RNA interference, we discovered several differentially expressed genes that regulate lifespan. We also found a compensatory role for translational regulation, which offsets dampened expression of a large subset of transcriptionally downregulated genes. Furthermore, 3’ UTR editing and intron retention increase under dietary restriction and correlate with diminished translation, while trans-spliced genes are refractory to reduced translation efficiency compared to messages with the native 5’ UTR. Finally, we find that smg-6 and smg-7, which are genes governing selection and turnover of nonsense mediated decay targets, are required for increased lifespan under dietary restriction.

scholarly journals Post-Transcriptional Regulation of Viral RNA through Epitranscriptional Modification

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1129
Author(s):  
David G. Courtney
Keyword(s):  
Transcriptional Regulation ◽  
Viral Replication ◽  
Viral Rna ◽  
Rna Modifications ◽  
Exact Role ◽  
Dna Viruses ◽  
Wide Range ◽  
Cellular Mrnas ◽  
Post Transcriptional Regulation ◽  
Rna And Dna

The field of mRNA modifications has been steadily growing in recent years as technologies have improved and the importance of these residues became clear. However, a subfield has also arisen, specifically focused on how these modifications affect viral RNA, with the possibility that viruses can also be used as a model to best determine the role that these modifications play on cellular mRNAs. First, virologists focused on the most abundant internal mRNA modification, m6A, mapping this modification and elucidating its effects on the RNA of a wide range of RNA and DNA viruses. Next, less common RNA modifications including m5C, Nm and ac4C were investigated and also found to be present on viral RNA. It now appears that viral RNA is littered with a multitude of RNA modifications. In biological systems that are under constant evolutionary pressure to out compete both the host as well as newly arising viral mutants, it poses an interesting question about what evolutionary benefit these modifications provide as it seems evident, at least to this author, that these modifications have been selected for. In this review, I discuss how RNA modifications are identified on viral RNA and the roles that have now been uncovered for these modifications in regard to viral replication. Finally, I propose some interesting avenues of research that may shed further light on the exact role that these modifications play in viral replication.

scholarly journals phiC31 integrase for recombination mediated single copy insertion and genome manipulation in C. elegans

2020 ◽  
Author(s):  
Fang-Jung Yang ◽  
Chiao-Nung Chen ◽  
Tiffany Chang ◽  
Ting-Wei Cheng ◽  
Ni-Chen Chang ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Integration Method ◽  
Single Copy ◽  
Large Set ◽  
Reporter System ◽  
Phic31 Integrase ◽  
C Elegans ◽  
Cassette Exchange ◽  
Post Transcriptional Regulation ◽  
Genome Manipulation

AbstractC. elegans benefits from a large set of tools for genome manipulation. Yet, the insertion of large DNA constructs and the generation of inversions is still challenging. Here, we adapted the phiC31 integrase system for C. elegans. We generated an integrated phiC31 integrase expressing strain flanked by attP sites that also serves as a landing pad for integration of transgenes by recombination mediated cassette exchange (RCME). This strain is unc-119(-) so RMCE integrants can be produced simply by injection of a plasmid carrying attB sites flanking unc-119(+) and the gene(s) of interest. Additionally, phiC31 integrase is removed concomitantly with integration, eliminating the need to outcross away the integrase. Integrations are relatively easy to obtain for insert sizes up to ~15 kb. Taking advantage of this integration method we establish a dual color fluorescent operon reporter system to study post-transcriptional regulation of mRNA. Last we show that large chromosomal segments can be inverted using phiC31 integrase. Thus the phiC31 integrase system should be a useful addition to the C. elegans toolkit.

43-OR: Post-transcriptional Regulation of Slc16a1 mRNA Is Essential for Maintaining Normal ß-Cell Function

Diabetes ◽  
2019 ◽  
Vol 68 (Supplement 1) ◽  
pp. 43-OR
Author(s):  
DINA MOSTAFA ◽  
AKINORI TAKAHASHI ◽  
TADASHI YAMAMOTO
Keyword(s):  
Transcriptional Regulation ◽  
Cell Function ◽  
Post Transcriptional Regulation
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