Effect of mutations on the p53 IRES RNA structure: Implications for de-regulation of the synthesis of p53 isoforms

Grover, Richa; Arandkar, Sharathchandra; Ponnuswamy, Anand; Khan, Debjit; Das, Saumitra

doi:10.4161/rna.8.1.14260

Cited by 43 publications

(53 citation statements)

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“…p53-null H1299 cells were transfected with luciferase bicistronic constructs containing p53 1-251 RNA in the intercistronic region. 7,8,11 Control cells and glucose-starved cells were harvested 4, 8, 20 and 30 h post-transfection. There was a consistent increase in the relative IRES activity at all these time points following glucose deprivation, with a 1.7-fold increase in activity by 20 h (Figure 1a, H1299 panel and Supplementary Table S1).…”

Section: Resultsmentioning

confidence: 99%

“…1 p53 and its N-terminally truncated isoform Δ40p53 (also known as ΔN-p53 or p53/47) are translated by internal ribosome entry site (IRES)-mediated translation initiation from the same mRNA under different stress conditions that induce DNA damage, ionizing radiation and endoplasmic reticulum (ER) stress, oncogene-induced senescence and cancer. [2][3][4][5][6][7] Thus, p53 mRNA has a dual IRES structure. 8 For their function, these IRESs rely on IRES trans-acting factors (ITAFs).…”

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confidence: 99%

“…3 This PTB-mediated control of p53 IRESs has been recently found to play a role in the p53-fibrillarin-rRNA methylation network. 9,10 Single-nucleotide polymorphisms in p53 5'UTR 11 or in the coding region of p53 IRES 7,11,12 decreased p53 IRES activity by altering PTB, MDM2 or hnRNPC1/C2 interactions with this regulatory region in p53 mRNA. Annexin A2 and PTB-associated splicing factor (PSF) proteins, putative p53 ITAFs, interact with p53 IRESs ex vivo in a stress-induced manner, showing greater association with the IRESs on thapsigargin treatment.…”

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Reversible induction of translational isoforms of p53 in glucose deprivation

et al. 2015

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Tumor suppressor protein p53 is a master transcription regulator, indispensable for controlling several cellular pathways. Earlier work in our laboratory led to the identification of dual internal ribosome entry site (IRES) structure of p53 mRNA that regulates translation of full-length p53 and Δ40p53. IRES-mediated translation of both isoforms is enhanced under different stress conditions that induce DNA damage, ionizing radiation and endoplasmic reticulum stress, oncogene-induced senescence and cancer. In this study, we addressed nutrient-mediated translational regulation of p53 mRNA using glucose depletion. In cell lines, this nutrient-depletion stress relatively induced p53 IRES activities from bicistronic reporter constructs with concomitant increase in levels of p53 isoforms. Surprisingly, we found scaffold/matrix attachment region-binding protein 1 (SMAR1), a predominantly nuclear protein is abundant in the cytoplasm under glucose deprivation. Importantly under these conditions polypyrimidine-tractbinding protein, an established p53 ITAF did not show nuclear-cytoplasmic relocalization highlighting the novelty of SMAR1-mediated control in stress. In vivo studies in mice revealed starvation-induced increase in SMAR1, p53 and Δ40p53 levels that was reversible on dietary replenishment. SMAR1 associated with p53 IRES sequences ex vivo, with an increase in interaction on glucose starvation. RNAi-mediated-transient SMAR1 knockdown decreased p53 IRES activities in normal conditions and under glucose deprivation, this being reflected in changes in mRNAs in the p53 and Δ40p53 target genes involved in cell-cycle arrest, metabolism and apoptosis such as p21, TIGAR and Bax. This study provides a new physiological insight into the regulation of this critical tumor suppressor in nutrient starvation, also suggesting important functions of the p53 isoforms in these conditions as evident from the downstream transcriptional target activation. Cell Death and Differentiation (2015) 22, 1203-1218; doi:10.1038/cdd.2014.220; published online 27 February 2015 p53 is a master transcription factor and tumor suppressor. Apart from post-translational modifications of p53 and concomitant protein-protein interactions of p53 with diverse factors, translational control of p53 mRNA also plays an important role under stress conditions. 1 p53 and its N-terminally truncated isoform Δ40p53 (also known as ΔN-p53 or p53/47) are translated by internal ribosome entry site (IRES)-mediated translation initiation from the same mRNA under different stress conditions that induce DNA damage, ionizing radiation and endoplasmic reticulum (ER) stress, oncogene-induced senescence and cancer. [2][3][4][5][6][7] Thus, p53 mRNA has a dual IRES structure. 8 For their function, these IRESs rely on IRES trans-acting factors (ITAFs). Polypyrimidine-tract-binding protein (PTB) was shown to have differential affinity for the two IRESs of p53 and regulated p53 IRES functions by translocating from nucleus to cytoplasm on doxorubicin-induced DNA damage. 3 This PTB-med...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Reversible induction of translational isoforms of p53 in glucose deprivation

et al. 2015

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“…Numerous reagents, including protein nucleases (Walczak et al 1996;Grover et al 2011;Siegfried et al 2011), alkylating chemicals such as dimethyl sulfate (DMS) (Wells et al 2000;Tijerina et al 2007;Cordero et al 2012a), and hydroxyl radicals (Adilakshmi et al 2006;Das et al 2008;Ding et al 2012), have been leveraged to modify or cleave RNA in a structure-dependent manner. Protection of nucleotides from modification, typically signaling the formation of base pairs, can guide manual or automatic secondary structure inference (Mathews et al 2004;Mitra et al 2008;Vasa et al 2008).…”

Section: Introductionmentioning

confidence: 99%

High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states

Tian

Cordero

Kladwang

et al. 2014

RNA

101

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The three-dimensional conformations of noncoding RNAs underpin their biochemical functions but have largely eluded experimental characterization. Here, we report that integrating a classic mutation/rescue strategy with high-throughput chemical mapping enables rapid RNA structure inference with unusually strong validation. We revisit a 16S rRNA domain for which SHAPE (selective 2 ′ -hydroxyl acylation with primer extension) and limited mutational analysis suggested a conformational change between apo-and holo-ribosome conformations. Computational support estimates, data from alternative chemical probes, and mutate-and-map (M 2 ) experiments highlight issues of prior methodology and instead give a nearcrystallographic secondary structure. Systematic interrogation of single base pairs via a high-throughput mutation/rescue approach then permits incisive validation and refinement of the M 2 -based secondary structure. The data further uncover the functional conformation as an excited state (20 ± 10% population) accessible via a single-nucleotide register shift. These results correct an erroneous SHAPE inference of a ribosomal conformational change, expose critical limitations of conventional structure mapping methods, and illustrate practical steps for more incisively dissecting RNA dynamic structure landscapes.

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“…This indicates that the majority of human p53 mRNAs have a 145-nucleotide 5=-UTR sequence. This fragment has been demonstrated to contain the IRES sequence of the p53 5= UTR using dual-luciferase reporter assays (10,11), and the IRES sequence has been further confirmed by DNA regional deletions and structural analysis (10,30). Since the sequence (145 nucleotides) harboring the p53 IRES is shorter than the p53 5=-UTR sequence (192 nucleotides) used in Takagi et al's assay to pull down TCP80 and RHA (6), we first tested whether or not TCP80 and RHA can bind specifically to the p53 IRES in vitro (10).…”

Section: Resultsmentioning

confidence: 99%

Deregulation of Internal Ribosome Entry Site-Mediated p53 Translation in Cancer Cells with Defective p53 Response to DNA Damage

Halaby

Harris

Miskimins

et al. 2015

Molecular and Cellular Biology

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Synthesis of the p53 tumor suppressor and its subsequent activation following DNA damage are critical for its protection against tumorigenesis. We previously discovered an internal ribosome entry site (IRES) at the 5= untranslated region of the p53 mRNA. However, the connection between IRES-mediated p53 translation and p53's tumor suppressive function is unknown. In this study, we identified two p53 IRES trans-acting factors, translational control protein 80 (TCP80), and RNA helicase A (RHA), which positively regulate p53 IRES activity. Overexpression of TCP80 and RHA also leads to increased expression and synthesis of p53. Furthermore, we discovered two breast cancer cell lines that retain wild-type p53 but exhibit defective p53 induction and synthesis following DNA damage. The levels of TCP80 and RHA are extremely low in both cell lines, and expression of both proteins is required to significantly increase the p53 IRES activity in these cells. Moreover, we found cancer cells transfected with a shRNA against TCP80 not only exhibit decreased expression of TCP80 and RHA but also display defective p53 induction and diminished ability to induce senescence following DNA damage. Therefore, our findings reveal a novel mechanism of p53 inactivation that links deregulation of IRES-mediated p53 translation with tumorigenesis.O ne of the most important tumor suppressors identified thus far is p53. Under normal conditions, p53 is inactive, and its cellular levels are low. In response to various genotoxic or cytotoxic stress, including DNA damage, p53 becomes activated (1). The activated p53 causes cell growth arrest or apoptosis, allowing damaged cells to self-repair or be eliminated. In this manner, p53 protects against malignant transformation of normal cells (2, 3). Activation of p53 involves both accumulation and posttranslational modification. It is thought that the control of p53 induction or accumulation occurs mainly at the translational and posttranslational levels (1). Although levels of p53 are known to be regulated by the ubiquitin ligase mouse double minute 2 (MDM2), now there is clear evidence showing that enhanced p53 translation is essential for its induction following DNA damage (4). More specifically, the 5= untranslated region (5= UTR) of p53 mRNA was found to be a major site for regulation of p53 translation (5, 6). The mechanism underlying translational regulation of p53 induction via its 5= UTR has started to emerge.Cap-dependent initiation of protein translation is used by the majority of mRNAs, since almost all eukaryotic mRNAs have an N 7 -methylguanosine cap structure at their 5= ends. Eukaryotic translation initiation factor 4E (eIF-4E), a translation initiation protein that binds to the cap structure, initiates the process (7). In situations where cap-dependent translation is impaired, including apoptosis or DNA damage, cap-independent protein translation, mediated by an internal ribosomal entry site (IRES) that does not require the participation of eIF-4E, is needed in eukaryotes for the synthe...

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Effect of mutations on the p53 IRES RNA structure: Implications for de-regulation of the synthesis of p53 isoforms

Cited by 43 publications

References 0 publications

Reversible induction of translational isoforms of p53 in glucose deprivation

Reversible induction of translational isoforms of p53 in glucose deprivation

High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states

Deregulation of Internal Ribosome Entry Site-Mediated p53 Translation in Cancer Cells with Defective p53 Response to DNA Damage

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