Virus-induced multiple gene silencing to study redundant metabolic pathways in plants: Silencing the starch degradation pathway in Nicotiana benthamiana

Context: Chronic myeloid leukemia (CML) is characterized by the presence of a fusion oncoprotein BCR-ABL. This mutation imparts a constitutive phosphorylation activity of tyrosine residues in the cellular proteins. One of the targets of BCR-ABL is the STAT5 protein, which when phosphorylated induces gene expression of antiapoptotic proteins such as BCL-XL. The STAT pathway has been targeted in the past by disrupting any one protein only. A multiple gene silencing has never been done in this pathway. Aim: The aim of this study was to compare the effects of downregulation of BCR-ABL, STAT5A, STAT5B, and BCL-XL, individually and simultaneously, in human CML cell line (K562 cells) through RNA interference (RNAi). Further, gene expression, inhibition of proliferation, and apoptosis induction were assessed in K562 cells. Materials and Methods: K562 cells were transfected with various combinations of small iRNA (siRNA) and the expressions of aforesaid genes were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. K562 cell proliferation and apoptosis were analyzed using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and flow cytometry, respectively. The results were compared through one-way analysis of variance. Results: qPCR and western blotting results post-siRNA transfection confirmed the targeted gene suppression and protein reduction in K562 cells. The cell proliferation assay and apoptosis assay revealed that simultaneous gene silencing of BCR-ABL, STAT5A, STAT5B, and BCL-XL had the highest killing effect on K562 cells as compared to knocking down these genes individually or in any other combinations. Conclusions: This was the first time it was shown that multiple gene silencing in STAT pathway in CML cell line K562 was better as compared to individual gene silencing.

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DDX21 interacts with nuclear AGO2 and regulates the alternative splicing of SMN2

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbaa029 ◽

2020 ◽

Vol 85 (2) ◽

pp. 272-279

Author(s):

Mengting Gong ◽

Xi Zhang ◽

Yaru Wang ◽

Guiyan Mao ◽

Yangqi Ou ◽

...

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Gene Silencing ◽

Target Gene ◽

Molecular Mechanisms ◽

Regulation Of Gene Expression ◽

Dependent Manner ◽

Indirect Interaction ◽

Multiple Gene ◽

Multiple Gene Silencing

ABSTRACT AGO2 is the only member of mammalian Ago protein family that possesses the catalytic activity and plays a central role in gene silencing. Recently researches reported that multiple gene silencing factors, including AGO2, function in the nuclei. The molecular mechanisms of the gene silencing factors functioning in nuclei are conducive to comprehend the roles of gene silencing in pretranslational regulation of gene expression. Here, we report that AGO2 interacts with DDX21 indirectly in an RNA-dependent manner by Co-IP and GST-Pulldown assays and the 2 proteins present nuclei foci in the immunofluorescence experiments. We found that DDX21 up-regulated the protein level of AGO2 and participated in target gene, SNM2, alternative splicing involved in AGO2 by the indirect interaction with AGO2, which produced different transcripts of SMN2 in discrepant expression level. This study laid important experiment foundation for the further analysis of the nuclear functions of gene silencing components.

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Involvement of Multiple Gene-Silencing Pathways in a Paramutation-like Phenomenon in Arabidopsis

Cell Reports ◽

10.1016/j.celrep.2015.04.034 ◽

2015 ◽

Vol 11 (8) ◽

pp. 1160-1167 ◽

Cited By ~ 7

Author(s):

Zhimin Zheng ◽

Hasi Yu ◽

Daisuke Miki ◽

Dan Jin ◽

Qingzhu Zhang ◽

...

Keyword(s):

Gene Silencing ◽

Multiple Gene ◽

Silencing Pathways ◽

Multiple Gene Silencing

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Multiple-Gene Silencing Using Antisense RNAs in Escherichia coli

Methods in Molecular Biology - Functional Genomics ◽

10.1007/978-1-61779-424-7_23 ◽

2011 ◽

pp. 307-319 ◽

Cited By ~ 13

Author(s):

Nobutaka Nakashima ◽

Shan Goh ◽

Liam Good ◽

Tomohiro Tamura

Keyword(s):

Escherichia Coli ◽

Gene Silencing ◽

Multiple Gene ◽

Antisense Rnas ◽

Multiple Gene Silencing

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Aberrant promoter methylation occurred from multicopy transgene and SU(VAR)3 - 9 homolog 9 (SUVH9) gene in transgenic Nicotiana benthamiana

Functional Plant Biology ◽

10.1071/fp12051 ◽

2012 ◽

Vol 39 (9) ◽

pp. 764 ◽

Cited By ~ 6

Author(s):

Gi-Ho Lee ◽

Seong-Han Sohn ◽

Eun-Young Park ◽

Young-Doo Park

Keyword(s):

Gene Silencing ◽

Nicotiana Benthamiana ◽

Fluorescent Protein ◽

Cauliflower Mosaic Virus ◽

Camv 35S Promoter ◽

35S Promoter ◽

Camv 35S ◽

Histone Deacetylase 1 ◽

Transgenic Lines ◽

Green Fluorescent

The chemical modification of DNA by methylation is a heritable trait and can be subsequently reversed without altering the original DNA sequence. Methylation can reduce or silence gene expression and is a component of a host’s defence response to foreign nucleic acids. In our study, we employed a plant transformation strategy using Nicotiana benthamiana Domin to study the heritable stability of the introduced transgenes. Through the introduction of the cauliflower mosaic virus (CaMV) 35S promoter and the green fluorescent protein (GFP) reporter gene, we demonstrated that this introduced promoter often triggers a homology-dependent gene-silencing (HDGS) response. These spontaneous transgene-silencing phenomena are due to methylation of the CaMV 35S promoter CAAT box during transgenic plant growth. This process is catalysed by SU(VAR)3–9 homologue 9 (SUVH9), histone deacetylase 1 (HDA1) and domains rearranged methylase 2 (DRM2). In particular, we showed from our data that SUVH9 is the key regulator of methylation activity in epigenetically silenced GFP transgenic lines; therefore, our findings demonstrate that an introduced viral promoter and transgene can be subject to a homology-dependent gene-silencing mechanism that can downregulate its expression and negatively influence the heritable stability of the transgene.

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High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

BMC Biotechnology ◽

10.1186/1472-6750-9-96 ◽

2009 ◽

Vol 9 (1) ◽

pp. 96 ◽

Cited By ~ 38

Author(s):

Raffaele Lombardi ◽

Patrizia Circelli ◽

Maria Villani ◽

Giampaolo Buriani ◽

Luca Nardi ◽

...

Keyword(s):

Gene Silencing ◽

Transient Expression ◽

Nicotiana Benthamiana ◽

Silencing Suppressor ◽

High Level ◽

Hiv 1

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Unusual Starch Degradation Pathway via Cyclodextrins in the Hyperthermophilic Sulfate-Reducing Archaeon Archaeoglobus fulgidus Strain 7324

Journal of Bacteriology ◽

10.1128/jb.01136-07 ◽

2007 ◽

Vol 189 (24) ◽

pp. 8901-8913 ◽

Cited By ~ 19

Author(s):

Antje Labes ◽

Peter Schönheit

Keyword(s):

Degradation Pathway ◽

Archaeoglobus Fulgidus ◽

Starch Degradation ◽

Cyclodextrin Glucanotransferase ◽

Gene Encoding ◽

Thermococcus Litoralis ◽

Sulfate Reducing ◽

Combined Action ◽

Archaeoglobus Fulgidus Strain 7324 ◽

Modified Embden Meyerhof Pathway

ABSTRACT The hyperthermophilic archaeon Archaeoglobus fulgidus strain 7324 has been shown to grow on starch and sulfate and thus represents the first sulfate reducer able to degrade polymeric sugars. The enzymes involved in starch degradation to glucose 6-phosphate were studied. In extracts of starch-grown cells the activities of the classical starch degradation enzymes, α-amylase and amylopullulanase, could not be detected. Instead, evidence is presented here that A. fulgidus utilizes an unusual pathway of starch degradation involving cyclodextrins as intermediates. The pathway comprises the combined action of an extracellular cyclodextrin glucanotransferase (CGTase) converting starch to cyclodextrins and the intracellular conversion of cyclodextrins to glucose 6-phosphate via cyclodextrinase (CDase), maltodextrin phosphorylase (Mal-P), and phosphoglucomutase (PGM). These enzymes, which are all induced after growth on starch, were characterized. CGTase catalyzed the conversion of starch to mainly β-cyclodextrin. The gene encoding CGTase was cloned and sequenced and showed highest similarity to a glucanotransferase from Thermococcus litoralis. After transport of the cyclodextrins into the cell by a transport system to be defined, these molecules are linearized via a CDase, catalyzing exclusively the ring opening of the cyclodextrins to the respective maltooligodextrins. These are degraded by a Mal-P to glucose 1-phosphate. Finally, PGM catalyzes the conversion of glucose 1-phosphate to glucose 6-phosphate, which is further degraded to pyruvate via the modified Embden-Meyerhof pathway.

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