A redesigned TetR-aptamer system to control gene expression in Plasmodium falciparum

AbstractOne of the most powerful approaches to understanding gene function involves turning genes on and off at will and measuring the impact at the cellular or organismal level. This particularly applies to the cohort of essential genes where traditional gene knockouts are inviable. In Plasmodium falciparum, conditional control of gene expression has been achieved by using multi-component systems in which individual modules interact with each other to regulate DNA recombination, transcription or posttranscriptional processes. The recently devised TetR-DOZI aptamer system relies on the ligand-regulatable interaction of a protein module with synthetic RNA aptamers to control the translation of a target gene. This technique has been successfully employed to study essential genes in P. falciparum and involves the insertion of several aptamer copies into their 3’ untranslated regions (UTRs) which provide control over mRNA fate. However, aptamer repeats are prone to recombination and one or more copies can be lost from the system, resulting in a loss of control over target gene expression. We rectified this issue by redesigning the aptamer array to minimize recombination while preserving the control elements. As proof of concept, we compared the original and modified arrays for their ability to knock down the levels of a putative essential apicoplast protein (PF3D7_0815700) and demonstrated that the modified array is highly stable and efficient. This redesign will enhance the utility of a tool that is quickly becoming a favored strategy for genetic studies in P. falciparum.ImportanceMalaria elimination efforts have been repeatedly hindered by the evolution and spread of multidrug-resistant strains of Plasmodium falciparum. The absence of a commercially available vaccine emphasizes the need for a better understanding of Plasmodium biology in order to further translational research. This has been partly facilitated by targeted gene deletion strategies for the functional analysis of parasite genes. However, genes that are essential for parasite replication in erythrocytes are refractory to such methods, and require conditional knockdown or knockout approaches to dissect their function. One such approach is the TetR-DOZI system that employs multiple synthetic aptamers in the untranslated regions of target genes to control their expression in a tetracycline-dependent manner. Maintaining modified parasites with intact aptamer copies has been challenging since these repeats are frequently lost by recombination. By interspacing the aptamers with unique sequences, we created a stable genetic system that remains effective at controlling target gene expression.

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Redesigned TetR-Aptamer System To Control Gene Expression in Plasmodium falciparum

mSphere ◽

10.1128/msphere.00457-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Krithika Rajaram ◽

Hans B. Liu ◽

Sean T. Prigge

Keyword(s):

Gene Expression ◽

Plasmodium Falciparum ◽

Target Gene ◽

Essential Genes ◽

Untranslated Regions ◽

Dependent Manner ◽

Control Of Gene Expression ◽

Content Type ◽

Target Gene Expression ◽

The Impact

ABSTRACT One of the most powerful approaches to understanding gene function involves turning genes on and off at will and measuring the impact at the cellular or organismal level. This particularly applies to the cohort of essential genes where traditional gene knockouts are inviable. In Plasmodium falciparum, conditional control of gene expression has been achieved by using multicomponent systems in which individual modules interact with each other to regulate DNA recombination, transcription, or posttranscriptional processes. The recently devised TetR-DOZI aptamer system relies on the ligand-regulatable interaction of a protein module with synthetic RNA aptamers to control the translation of a target gene. This technique has been successfully employed to study essential genes in P. falciparum and involves the insertion of several aptamer copies into the 3′ untranslated regions (UTRs), which provide control over mRNA fate. However, aptamer repeats are prone to recombination and one or more copies can be lost from the system, resulting in a loss of control over target gene expression. We rectified this issue by redesigning the aptamer array to minimize recombination while preserving the control elements. As proof of concept, we compared the original and modified arrays for their ability to knock down the levels of a putative essential apicoplast protein (PF3D7_0815700) and demonstrated that the modified array is highly stable and efficient. This redesign will enhance the utility of a tool that is quickly becoming a favored strategy for genetic studies in P. falciparum. IMPORTANCE Malaria elimination efforts have been repeatedly hindered by the evolution and spread of multidrug-resistant strains of Plasmodium falciparum. The absence of a commercially available vaccine emphasizes the need for a better understanding of Plasmodium biology in order to further translational research. This has been partly facilitated by targeted gene deletion strategies for the functional analysis of parasite genes. However, genes that are essential for parasite replication in erythrocytes are refractory to such methods, and require conditional knockdown or knockout approaches to dissect their function. One such approach is the TetR-DOZI system that employs multiple synthetic aptamers in the untranslated regions of target genes to control their expression in a tetracycline-dependent manner. Maintaining modified parasites with intact aptamer copies has been challenging since these repeats can be lost by recombination. By interspacing the aptamers with unique sequences, we created a stable genetic system that remains effective at controlling target gene expression.

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Loss of miR-83 extends lifespan and affects target gene expression in an age-dependent manner in Caenorhabditis elegans

Journal of Genetics and Genomics ◽

10.1016/j.jgg.2018.11.003 ◽

2018 ◽

Vol 45 (12) ◽

pp. 651-662 ◽

Cited By ~ 5

Author(s):

Emmanuel Enoch Dzakah ◽

Ahmed Waqas ◽

Shuai Wei ◽

Bin Yu ◽

Xiaolin Wang ◽

...

Keyword(s):

Gene Expression ◽

Caenorhabditis Elegans ◽

Target Gene ◽

Dependent Manner ◽

Target Gene Expression ◽

Age Dependent

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E2f1, E2f2, and E2f3 Control E2F Target Expression and Cellular Proliferation via a p53-Dependent Negative Feedback Loop

Molecular and Cellular Biology ◽

10.1128/mcb.02147-05 ◽

2007 ◽

Vol 27 (1) ◽

pp. 65-78 ◽

Cited By ~ 59

Author(s):

Cynthia Timmers ◽

Nidhi Sharma ◽

Rene Opavsky ◽

Baidehi Maiti ◽

Lizhao Wu ◽

...

Keyword(s):

Gene Expression ◽

Target Gene ◽

Target Genes ◽

Cellular Proliferation ◽

Kinase Activity ◽

Cyclin Dependent Kinase ◽

Control Of Gene Expression ◽

Target Gene Expression ◽

Rb Phosphorylation ◽

P53 Target Genes

ABSTRACT E2F-mediated control of gene expression is believed to have an essential role in the control of cellular proliferation. Using a conditional gene-targeting approach, we show that the targeted disruption of the entire E2F activator subclass composed of E2f1, E2f2, and E2f3 in mouse embryonic fibroblasts leads to the activation of p53 and the induction of p53 target genes, including p21 CIP1 . Consequently, cyclin-dependent kinase activity and retinoblastoma (Rb) phosphorylation are dramatically inhibited, leading to Rb/E2F-mediated repression of E2F target gene expression and a severe block in cellular proliferation. Inactivation of p53 in E2f1-, E2f2-, and E2f3-deficient cells, either by spontaneous mutation or by conditional gene ablation, prevented the induction of p21 CIP1 and many other p53 target genes. As a result, cyclin-dependent kinase activity, Rb phosphorylation, and E2F target gene expression were restored to nearly normal levels, rendering cells responsive to normal growth signals. These findings suggest that a critical function of the E2F1, E2F2, and E2F3 activators is in the control of a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation.

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MiR-124 synergism with ELAVL3 enhances target gene expression to promote neuronal maturity

10.1101/2020.03.25.006635 ◽

2020 ◽

Author(s):

Ya-Lin Lu ◽

Yangjian Liu ◽

Matthew J. McCoy ◽

Andrew S. Yoo

Keyword(s):

Gene Expression ◽

Mirna Target ◽

Target Gene ◽

Target Genes ◽

Dependent Manner ◽

Neuronal Function ◽

Mirna Target Gene ◽

Target Gene Expression ◽

Human Neurons

SummaryNeuron-enriched microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), direct cell fate switching of human fibroblasts to neurons when ectopically expressed by repressing anti-neurogenic genes. How these miRNAs function after the onset of the transcriptome switch to a neuronal fate remains unclear. Here, we identified direct targets of miRNAs by Argonaute (AGO) HITS-CLIP as reprogramming cells activate the neuronal program and reveal the role of miR-124 that directly promotes the expression of its target genes associated with neuronal development and function. The mode of miR-124 as a positive regulator is determined by a neuron-enriched RNA-binding protein, ELAVL3, that interacts with AGO and binds target transcripts, whereas the non-neuronal ELAVL1 counterpart fails to elevate the miRNA-target gene expression. Although existing literature indicate that miRNA-ELAVL1 interaction can result in either target gene upregulation or downregulation in a context-dependent manner, we specifically identified neuronal ELAVL3 as the driver for miRNA target gene upregulation in neurons. In primary human neurons, repressing miR-124 and ELAVL3 led to the downregulation of genes involved in neuronal function and process outgrowth, and cellular phenotypes of reduced inward currents and neurite outgrowth. Results from our study support the role of miR-124 promoting neuronal function through positive regulation of its target genes.

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A tight cold-inducible switch built by coupling thermosensitive transcriptional and proteolytic regulatory parts

Nucleic Acids Research ◽

10.1093/nar/gkz785 ◽

2019 ◽

Vol 47 (21) ◽

pp. e137-e137 ◽

Cited By ~ 2

Author(s):

Yang Zheng ◽

Fankang Meng ◽

Zihui Zhu ◽

Weijia Wei ◽

Zhi Sun ◽

...

Keyword(s):

Gene Expression ◽

High Performance ◽

Target Gene ◽

Dynamic Range ◽

Culture Media ◽

Basic Research ◽

Control Of Gene Expression ◽

Target Gene Expression ◽

Engineered Systems ◽

Bacterial Genes

Abstract Natural organisms have evolved intricate regulatory mechanisms that sense and respond to fluctuating environmental temperatures in a heat- or cold-inducible fashion. Unlike dominant heat-inducible switches, very few cold-inducible genetic switches are available in either natural or engineered systems. Moreover, the available cold-inducible switches still have many shortcomings, including high leaky gene expression, small dynamic range (<10-fold) or broad transition temperature (>10°C). To address these problems, a high-performance cold-inducible switch that can tightly control target gene expression is highly desired. Here, we introduce a tight and fast cold-inducible switch that couples two evolved thermosensitive variants, TFts and TEVts, as well as an additional Mycoplasma florum Lon protease (mf-Lon) to effectively turn-off target gene expression via transcriptional and proteolytic mechanisms. We validated the function of the switch in different culture media and various Escherichia coli strains and demonstrated its tightness by regulating two morphogenetic bacterial genes and expressing three heat-unstable recombinant proteins, respectively. Moreover, the additional protease module enabled the cold-inducible switch to actively remove the pre-existing proteins in slow-growing cells. This work establishes a high-performance cold-inducible system for tight and fast control of gene expression which has great potential for basic research, as well as industrial and biomedical applications.

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p53 Pulses Diversify Target Gene Expression Dynamics in an mRNA Half-Life-Dependent Manner and Delineate Co-regulated Target Gene Subnetworks

Cell Systems ◽

10.1016/j.cels.2016.03.006 ◽

2016 ◽

Vol 2 (4) ◽

pp. 272-282 ◽

Cited By ~ 41

Author(s):

Joshua R. Porter ◽

Brian E. Fisher ◽

Eric Batchelor

Keyword(s):

Gene Expression ◽

Half Life ◽

Target Gene ◽

Dependent Manner ◽

Target Gene Expression ◽

Gene Expression Dynamics ◽

Mrna Half Life

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Methodological considerations on selection of stable reference genes for RT-qPCR in the neonatal rat brain in hypoxia and hypothermia

10.1101/793786 ◽

2019 ◽

Author(s):

M. Bustelo ◽

M.A. Bruno ◽

C.F. Loidl ◽

H.W.M. Steinbusch ◽

A.W.D. Gavilanes ◽

...

Keyword(s):

Gene Expression ◽

Rat Brain ◽

Statistical Methods ◽

Target Gene ◽

Expression Profiles ◽

Neonatal Rat ◽

Experimental Conditions ◽

Target Gene Expression ◽

The Impact ◽

Neonatal Rat Brain

AbstractReal-time reverse transcription PCR (qPCR) normalized to an internal reference gene (RG), is a frequently used method for quantifying gene expression changes in neuroscience. Although RG expression is assumed to be constantly independent of physiological or experimental conditions, several studies have shown that commonly used RGs are not expressed stably. The use of unstable RGs has a profound effect on the conclusions drawn from studies on gene expression, and almost universally results in spurious estimation of target gene expression. Approaches aimed at selecting and validating RGs often make use of different statistical methods, which may lead to conflicting results. The present study evaluates the expression of 5 candidate RGs (Actb, Pgk1, Sdha, Gapdh, Rnu6b) as a function of hypoxia exposure and hypothermic treatment in the neonatal rat cerebral cortex –in order to identify RGs that are stably expressed under these experimental conditions– and compares several statistical approaches that have been proposed to validate RGs. In doing so, we first analyzed the RG ranking stability proposed by several widely used statistical methods and related tools, i.e. the Coefficient of Variation (CV) analysis, GeNorm, NormFinder, BestKeeper, and the ΔCt method. Subsequently, we compared RG expression patterns between the various experimental groups. We found that these statistical methods, next to producing different rankings per se, all ranked RGs displaying significant differences in expression levels between groups as the most stable RG. As a consequence, when assessing the impact of RG selection on target gene expression quantification, substantial differences in target gene expression profiles were observed. As such, by assessing mRNA expression profiles within the neonatal rat brain cortex in hypoxia and hypothermia as a showcase, this study underlines the importance of further validating RGs for each new experimental paradigm considering the limitations of each selection method.

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2049-P: The Chd4 Helicase of the Nucleosome Remodeling and Deacetylase Complex Modulates Pdx1 Target Gene Expression In Vitro

Diabetes ◽

10.2337/db20-2049-p ◽

2020 ◽

Vol 69 (Supplement 1) ◽

pp. 2049-P

Author(s):

REBECCA K. DAVIDSON ◽

NOLAN CASEY ◽

JASON SPAETH

Keyword(s):

Gene Expression ◽

Target Gene ◽

Nucleosome Remodeling ◽

Target Gene Expression

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On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11269-z ◽

2021 ◽

Author(s):

Philipp Moritz Fricke ◽

Angelika Klemm ◽

Michael Bott ◽

Tino Polen

Keyword(s):

Gene Expression ◽

Acetic Acid ◽

Literature Search ◽

Target Gene ◽

Target Genes ◽

Recombinant Dna ◽

Acetic Acid Bacteria ◽

Homoserine Lactone ◽

Expression Systems ◽

Target Gene Expression

Abstract Acetic acid bacteria (AAB) are valuable biocatalysts for which there is growing interest in understanding their basics including physiology and biochemistry. This is accompanied by growing demands for metabolic engineering of AAB to take advantage of their properties and to improve their biomanufacturing efficiencies. Controlled expression of target genes is key to fundamental and applied microbiological research. In order to get an overview of expression systems and their applications in AAB, we carried out a comprehensive literature search using the Web of Science Core Collection database. The Acetobacteraceae family currently comprises 49 genera. We found overall 6097 publications related to one or more AAB genera since 1973, when the first successful recombinant DNA experiments in Escherichia coli have been published. The use of plasmids in AAB began in 1985 and till today was reported for only nine out of the 49 AAB genera currently described. We found at least five major expression plasmid lineages and a multitude of further expression plasmids, almost all enabling only constitutive target gene expression. Only recently, two regulatable expression systems became available for AAB, an N-acyl homoserine lactone (AHL)-inducible system for Komagataeibacter rhaeticus and an l-arabinose-inducible system for Gluconobacter oxydans. Thus, after 35 years of constitutive target gene expression in AAB, we now have the first regulatable expression systems for AAB in hand and further regulatable expression systems for AAB can be expected. Key points • Literature search revealed developments and usage of expression systems in AAB. • Only recently 2 regulatable plasmid systems became available for only 2 AAB genera. • Further regulatable expression systems for AAB are in sight.

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