Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5′-Terminal Region of p53 mRNA In Vitro and in the Living Cells

Górska, Agnieszka; Świątkowska, Agata; Dutkiewicz, Mariola; Ciesiołka, Jerzy

doi:10.1371/journal.pone.0078863

Cited by 12 publications

(26 citation statements)

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“…Both mapping patterns were compared with the pattern that has been earlier obtained for the corresponding region in human p53 mRNA. It turned out that the presence of a single hairpin motif in that region is more consistent with the rules that govern hybridization of complementary oligonucleotides [35] and the mapping pattern characteristic of the 5′-terminal region of human p53 mRNA [36]. Therefore, this region seems to form a single hairpin motif in both mouse and human p53 mRNAs, which in human transcript has been shown to interact with Mdm2 protein [37].…”

Section: Secondary Structure Of Rna-122mentioning

confidence: 56%

“…This approach has been shown to be particularly useful in screening for target sites for antisense oligonucleotides in highly structured RNA molecules: region X of hepatitis C virus [34], and the genomic and antigenomic HDV ribozymes [35]. Most importantly, this approach was used for mapping the sites accessible to oligomer hybridization in the 5′-terminal region of human p53 mRNA that begins at P1 transcription initiation site [36]. This data allowed us to make direct comparison of accessibility to hybridization of both the corresponding regions of mouse and human p53 mRNAs.…”

Section: Secondary Structure Of Rna-122mentioning

confidence: 99%

“…Mapping of RNA accessibility to hybridization with DNA semi-random libraries and RNase H cleavage Mapping of the RNA fragment corresponding to the G33 -A89 region was conducted based on the previous work [35,36]. Briefly, 0.5 pmol mRNA-122 was incubated in the buffer: 20 mM HEPES pH 8.0, 50 mM KCl, 4 mM MgCl 2 , 1 mM DTT and 50 μg/ml BSA at 65°C for 2 min and cooled to 37°C.…”

Section: Flow Cytometrymentioning

confidence: 99%

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Length and secondary structure of the 5′ non-coding regions of mouse p53 mRNA transcripts - mouse as a model organism for p53 gene expression studies

2018

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Transcription initiation sites of Trp53 gene in mice were determined using the 5′RACE method. Based on sequence alignment of the 5′-terminal regions of p53 mRNA in mammals, the site for the most abundant transcript turned out to be essentially identical with that determined for human TP53 gene and slightly differed for the longest transcripts, in mice and humans. Secondary structures of the 5′-terminal regions of the shorter, most abundant and the longest mouse transcripts were determined in vitro and the shorter transcript was also mapped in transfected mouse cells. For the first time, secondary structure models of the 5′ terminus of two mouse p53 mRNAs were proposed. Comparing these models with the conservativeness of the nucleotide sequence of the 5′-terminal region of mRNA in mouse and other mammals, the possible function of the selected structural domains of this region was discussed. To elucidate the translation mechanisms, the two studied mRNAs were translated in the presence of an increasing concentration of the cap analog. For the longest transcript, the data suggested that IRES element(s) was/were involved in translation initiation. Additionally, changes in p53 synthesis under genotoxic and endoplasmic reticulum stress conditions in mouse cells were analyzed.

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Section: Secondary Structure Of Rna-122mentioning

confidence: 56%

Section: Secondary Structure Of Rna-122mentioning

confidence: 99%

Section: Flow Cytometrymentioning

confidence: 99%

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Length and secondary structure of the 5′ non-coding regions of mouse p53 mRNA transcripts - mouse as a model organism for p53 gene expression studies

2018

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“…A possible mechanism of an auto-regulatory feedback loop (right). tious diseases caused by such dangerous viruses as HIV or HCV (Figlerowicz et al, 2003;Kurzynska-Kokorniak et al, 2009;Miazga et al, 2011;Jackowiak et al, 2012;Gorska et al, 2013;Jackowiak et al, 2014;Dutkiewicz et al, 2015;Belter et al, 2016).…”

Section: Different Mechanisms Of the Hdicer Activity Regulation By Shmentioning

confidence: 99%

How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing

et al. 2017

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Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by a variety of proteins are being elucidated, less is known about non-protein factors, e.g. RNA molecules, that may influence this enzyme's activity. Therefore, we decided to investigate the question of whether the RNA molecules can function not only as Dicer substrates but also as its regulators. Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript, we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs that are longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding to this enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by participating in regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.

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“…LNA longRNA GapmeRs were ordered from Exiqon. The control gapmer ( ACCagggcgtatctctccATA ) [103] and p21-specific gapmer ( TCCgcgcccagCTCC ) [77] were administered to cells via gymnosis. The uppercase letters indicate the LNA while the lower case letters indicate phosphorothioated bases.…”

Section: Methodsmentioning

confidence: 99%

Epstein-Barr Virus Nuclear Antigen 3A Promotes Cellular Proliferation by Repression of the Cyclin-Dependent Kinase Inhibitor p21WAF1/CIP1

Tursiella

Bowman²,

Wanzeck³

et al. 2014

PLoS Pathog

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Latent infection by Epstein-Barr virus (EBV) is highly associated with the endemic form of Burkitt lymphoma (eBL), which typically limits expression of EBV proteins to EBNA-1 (Latency I). Interestingly, a subset of eBLs maintain a variant program of EBV latency - Wp-restricted latency (Wp-R) - that includes expression of the EBNA-3 proteins (3A, 3B and 3C), in addition to EBNA-1. In xenograft assays, Wp-R BL cell lines were notably more tumorigenic than their counterparts that maintain Latency I, suggesting that the additional latency-associated proteins expressed in Wp-R influence cell proliferation and/or survival. Here, we evaluated the contribution of EBNA-3A. Consistent with the enhanced tumorigenic potential of Wp-R BLs, knockdown of EBNA-3A expression resulted in abrupt cell-cycle arrest in G0/G1 that was concomitant with conversion of retinoblastoma protein (Rb) to its hypophosphorylated state, followed by a loss of Rb protein. Comparable results were seen in EBV-immortalized B lymphoblastoid cell lines (LCLs), consistent with the previous observation that EBNA-3A is essential for sustained growth of these cells. In agreement with the known ability of EBNA-3A and EBNA-3C to cooperatively repress p14ARF and p16INK4a expression, knockdown of EBNA-3A in LCLs resulted in rapid elevation of p14ARF and p16INK4a. By contrast, p16INK4a was not detectably expressed in Wp-R BL and the low-level expression of p14ARF was unchanged by EBNA-3A knockdown. Amongst other G1/S regulatory proteins, only p21WAF1/CIP1, a potent inducer of G1 arrest, was upregulated following knockdown of EBNA-3A in Wp-R BL Sal cells and LCLs, coincident with hypophosphorylation and destabilization of Rb and growth arrest. Furthermore, knockdown of p21WAF1/CIP1 expression in Wp-R BL correlated with an increase in cellular proliferation. This novel function of EBNA-3A is distinct from the functions previously described that are shared with EBNA-3C, and likely contributes to the proliferation of Wp-R BL cells and LCLs.

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Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5′-Terminal Region of p53 mRNA In Vitro and in the Living Cells

Cited by 12 publications

References 44 publications

Length and secondary structure of the 5′ non-coding regions of mouse p53 mRNA transcripts - mouse as a model organism for p53 gene expression studies

Length and secondary structure of the 5′ non-coding regions of mouse p53 mRNA transcripts - mouse as a model organism for p53 gene expression studies

How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing

Epstein-Barr Virus Nuclear Antigen 3A Promotes Cellular Proliferation by Repression of the Cyclin-Dependent Kinase Inhibitor p21WAF1/CIP1

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