A dual luciferase system for analysis of post-transcriptional regulation of gene expression in Leishmania

Soysa, Radika; Carter, Nicola S.; Yates, Phillip A.

doi:10.1016/j.molbiopara.2014.05.002

Cited by 13 publications

(27 citation statements)

References 20 publications

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“…Posttranscriptional regulation of proteins primarily involves altering protein stability through posttranslational modification, such as ubiquitination, N ‐glycosylation, phosphorylation, or sumoylation (26). Turnover rates of α‐actinin‐4 were measured in COS7 cells treated with the protein synthesis inhibitor cycloheximide (CHX).…”

Section: Resultsmentioning

confidence: 99%

NHERF1 regulates actin cytoskeleton organization through modulation of α‐actinin‐4 stability

et al. 2015

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The actin cytoskeleton is composed of a highly dynamic network of filamentous proteins, yet the molecular mechanism that regulates its organization and remodeling remains elusive. In this study, Na + /H + exchanger regulatory factor (NHERF)-1 loss-of-function and gain-of-function experiments reveal that polymerized actin cytoskeleton (F-actin) in HeLa cells is disorganized by NHERF1, whereas actin protein expression levels exhibit no detectable change. To elucidate the molecular mechanism underlying actin cytoskeleton disorganization by NHERF1, a combined 2-dimensional electrophoresismatrix-assisted laser desorption/ionization-time of flight mass spectrometry approach was used to screen for proteins regulated by NHERF1 in HeLa cells. a-Actinin-4, an actin cross-linking protein, was identified. Glutathione S-transferase pull-down and coimmunoprecipitation studies showed the a-actinin-4 carboxyl-terminal region specifically interacted with the NHERF1 postsynaptic density 95/disc-large/zona occludens-1 domain. The NHERF1/ a-actinin-4 interaction increased a-actinin-4 ubiquitination and decreased its expression levels, resulting in actin cytoskeleton disassembly. Our study identified a-actinin-4 as a novel NHERF1 interaction partner and provided new insights into the regulatory mechanism of the actin cytoskeleton by NHERF1

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Section: Resultsmentioning

confidence: 99%

NHERF1 regulates actin cytoskeleton organization through modulation of α‐actinin‐4 stability

et al. 2015

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“…cn/-/media/files/resources/protocols/technical-manuals/0/dualluciferase-reporter-assay-system-protocol.pdf?la=zh-cn; Soysa et al, 2014). cn/-/media/files/resources/protocols/technical-manuals/0/dualluciferase-reporter-assay-system-protocol.pdf?la=zh-cn; Soysa et al, 2014).…”

Section: Transfection Assaymentioning

confidence: 99%

“…The cells were harvested 48 h after co-transfection, and the luciferase activity was measured as the ratio of Firefly/Renilla activity using a Dual-luciferase assay system, according to the manufacturer's protocol (E1910; Promega), for which good stability has been previously verified (https://www.promega.com. cn/-/media/files/resources/protocols/technical-manuals/0/dualluciferase-reporter-assay-system-protocol.pdf?la=zh-cn; Soysa et al, 2014).…”

Section: Transfection Assaymentioning

confidence: 99%

Regulation of insulin resistance by targeting the insulin‐like growth factor 1 receptor with microRNA‐122‐5p in hepatic cells

Dong

Hou

Liu

et al. 2019

Cell Biology International

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Insulin resistance (IR) is a common etiology of type 2 diabetes (T2D) defined by a state of decreased reactivity to insulin in multiple organs, such as the liver. This study aims to investigate how microRNA-122-5p (miR-122) regulates the hepatic IR in vitro. We first found that the miR-122 level was upregulated in the liver of rats fed with a high-fat diet and injected with streptozotocin (T2D rats), while the expression level of insulin-like growth factor 1 receptor (IGF-1R), a potential target of miR-122, was downregulated in the diabetic liver. In vitro, glucosamine-induced IR was introduced in HepG2 hepatic cells, and the levels of miR-122 and IGF-1R were further assessed. An increase of miR-122 level and a decrease of IGF-IR level were observed in IR hepatic cells, which was the same as that in the diabetic liver. Results of the luciferase reporter assay validated IGF-1R as a direct target of miR-122. Moreover, in IR HepG2 cells, antagonizing miR-122 with its specific inhibitor enhanced glucose uptake and suppressed the expression of glucose 6-phosphatase and phosphoenolpyruvate carboxykinase, two key enzymes in regulating gluconeogenesis. Such alterations induced by the miR-122 inhibitor in IR hepatic cells were impaired when IGF-1R was simultaneously knocked down. In addition, the PI3K/Akt pathway was deactivated in IR cells, and then reactivated with miR-122 inhibitor transfection. In conclusion, our study demonstrates that miR-122 is able to regulate IR in hepatic cells by targeting IGF-1R.

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“…The L. donovani purine nucleobase transporter 3 (LdNT3) is one of the earliest and most substantially upregulated proteins in purine-starved L. donovani. LdNT3 upregulation is mediated in part at the levels of mRNA abundance and translational efficiency (16,18). Here we demonstrate that LdNT3 protein stability is not altered under purine stress, establishing the dominance of mRNA-level and translational control points in purine-responsive LdNT3 regulation.…”

mentioning

confidence: 56%

Purine-responsive expression of theLeishmania donovaniNT3 purine nucleobase transporter is mediated by a conserved RNA stem-loop

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Yates

2020

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ABSTRACTThe ability to modulate gene expression in response to changes in the host environment is essential for survival of the kinetoplastid parasite Leishmania. Unlike most eukaryotes, gene expression in kinetoplastids is predominately regulated post-transcriptionally. Consequently, RNA-binding proteins (RBPs) and mRNA-encoded sequence elements serve as primary determinants of gene regulation in these organisms; however, few have been ascribed roles in specific stress-response pathways. Leishmania lack the capacity for de novo purine synthesis and must scavenge these essential nutrients from the host. Leishmania have evolved a robust stress response to withstand sustained periods of purine scarcity during their lifecycle. The purine nucleobase transporter, LdNT3, is among the most substantially upregulated proteins in purine-starved L. donovani. Here we report that the post-translational stability of the LdNT3 protein is unchanged in response to purine starvation. Instead, LdNT3 upregulation is primarily mediated by a 33 nucleotide (nt) sequence in the LdNT3 mRNA 3’-untranslated region that is predicted to adopt a stem-loop structure. While this sequence is highly conserved within the mRNAs of orthologous transporters in multiple kinetoplastid species, putative stem-loops from L. donovani and Trypanosoma brucei nucleobase transporter mRNAs are not functionally interchangeable for purine-responsive regulation. Through mutational analysis of the element, we demonstrate that species specificity is attributable to just three variant bases within the predicted loop. Finally, we provide evidence that the abundance of the trans-acting factor that binds the LdNT3 stem-loop in vivo is substantially higher than required for regulation of LdNT3 alone, implying a potential role in regulating other purine-responsive genes.

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A dual luciferase system for analysis of post-transcriptional regulation of gene expression in Leishmania

Cited by 13 publications

References 20 publications

NHERF1 regulates actin cytoskeleton organization through modulation of α‐actinin‐4 stability

NHERF1 regulates actin cytoskeleton organization through modulation of α‐actinin‐4 stability

Regulation of insulin resistance by targeting the insulin‐like growth factor 1 receptor with microRNA‐122‐5p in hepatic cells

Purine-responsive expression of theLeishmania donovaniNT3 purine nucleobase transporter is mediated by a conserved RNA stem-loop

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