P53 mRNA Translation Research Articles

P53 and HuR combinatorially control the biphasic dynamics of microRNA-125b in response to genotoxic stress

Post-transcriptional regulation of p53, by the microRNA miR-125b and the RNA-binding protein HuR, controls p53 expression under genotoxic stress. p53 mRNA translation is repressed by miR-125b, tightly regulating its basal level of expression. The repression is relieved upon DNA damage by a decrease in miR-125b level, contributing to pulsatile expression of p53. The pulse of p53, as also of HuR, in response to UV irradiation coincides with a time-dependent biphasic change in miR-125b level. We show that the cause for the decrease in miR-125b level immediately post DNA-damage is enhanced exosomal export mediated by HuR. The subsequent increase in miR-125b level is due to p53-mediated transcriptional upregulation and enhanced processing, demonstrating miR-125b as a transcriptional and processing target of p53. p53 activates the transcription of primary miR-125b RNA from a cryptic promoter in response to UV irradiation. Together, these regulatory processes constitute reciprocal feedback loops that determine the biphasic change in miR-125b level, ultimately contributing to the fine-tuned temporal regulation of p53 expression in response to genotoxic stress.

Tumor Suppressor p53 Down-Regulates Programmed Cell Death Protein 4 (PDCD4) Expression.

The programmed cell death protein 4 (PDCD4), a well-known tumor suppressor, inhibits translation initiation and cap-dependent translation by inhibiting the helicase activity of EIF4A. The EIF4A tends to target mRNAs with a structured 5'-UTR. In addition, PDCD4 can also prevent tumorigenesis by inhibiting tumor promoter-induced neoplastic transformation, and studies indicate that PDCD4 binding to certain mRNAs inhibits those mRNAs' translation. A previous study demonstrated that PDCD4 inhibits the translation of p53 mRNA and that treatment with DNA-damaging agents down-regulates PDCD4 expression but activates p53 expression. The study further demonstrated that treatment with DNA-damaging agents resulted in the downregulation of PDCD4 expression and an increase in p53 expression, suggesting a potential mechanism by which p53 regulates the expression of PDCD4. However, whether p53 directly regulates PDCD4 remains unknown. Herein, we demonstrate for the first time that p53 regulates PDCD4 expression. Firstly, we found that overexpression of p53 in p53-null cells (H1299 and Saos2 cells) decreased the PDCD4 protein level. Secondly, p53 decreased PDCD4 promoter activity in gene reporter assays. Moreover, we demonstrated that mutations in p53 (R273H: contact hotspot mutation, and R175H: conformational hotspot mutation) abolished p53-mediated PDCD4 repression. Furthermore, mutations in the DNA-binding domain, but not in the C-terminal regulatory domain, of p53 disrupted p53-mediated PDCD4 repression. Finally, the C-terminal regulatory domain truncation study showed that the region between aa374 and aa370 is critical for p53-mediated PDCD4 repression. Taken together, our results suggest that p53 functions as a novel regulator of PDCD4, and the relationship between p53 and PDCD4 may be involved in tumor development and progression.

Optimization of eIF4E-Binding Peptide Pep8 to Disrupt the RBM38-eIF4E Complex for Induction of p53 and Tumor Suppression.

Interaction of RNA-binding protein RBM38 with eIF4E on p53 mRNA is known to suppress p53 mRNA translation, which can be disrupted by an 8-amino acid peptide (Pep8-YPYAASPA) derived from RBM38, leading to induction of p53 and tumor suppression. Here, we rationally designed multiple Pep8 derivatives and screened for their binding affinities towards eIF4E in silico. We showed that several key residues within Pep8 are necessary for its structure and function. We identified a shortened 7-amino acid peptide (Pep7-PSAASPV) that has the highest affinity towards eIF4E and is the most potent inducer of p53 expression. We found that iRGD is an effective vehicle to deliver Pep7 inside of cells for induction of p53 expression and growth suppression as compared to other cell penetrating peptides (Penetratin and Pep-1). We found that peptide cyclization enhances Pep8 affinity for eIF4E, induction of p53 and tumor cell growth suppression. We also found that the ability of Pep7 to induce p53 expression and growth suppression is conserved in cells derived from canine osteosarcoma, a spontaneous tumor model frequently used for testing the feasibility of a therapeutic agent for human cancer. Moreover, we showed that both human and canine osteosarcoma cells, which are notoriously resistant to radiation therapy, were sensitized by Pep7 to radiation-induced growth suppression and cell death. Together, our data suggest that Pep7 may be explored to sensitize tumors to radiation therapy.

Poly(C)-binding Protein 2 Regulates the p53 Expression via Interactions with the 5'-Terminal Region of p53 mRNA.

The p53 protein is one of the major transcriptional factors which guards cell homeostasis. Here, we showed that poly(C)-binding protein 2 (PCBP2) can bind directly to the 5′ terminus of p53 mRNA by means of electrophoretic mobility shift assay. Binding sites of PCBP2 within this region of p53 mRNA were mapped using Pb2+-induced cleavage and SAXS methods. Strikingly, the downregulation of PCBP2 in HCT116 cells resulted in a lower level of p53 protein under normal and stress conditions. Quantitative analysis of p53 mRNA in PCBP2-downregulated cells revealed a lower level of p53 mRNA under normal conditions suggesting the involvement of PCBP2 in p53 mRNA stabilisation. However, no significant change in p53 mRNA level was observed upon PCBP2 depletion under genotoxic stress. Moreover, a higher level of p53 protein in the presence of rapamycin or doxorubicin and the combination of both antibiotics was noticed in PCBP2-overexpressed cells compared to control cells. These observations indicate the potential involvement of PCBP2 in cap-independent translation of p53 mRNA especially occurring under stress conditions. It has been postulated that the PCBP2 protein is engaged in the enhancement of p53 mRNA stability, probably via interacting with its 3′ end. Our data show that under stress conditions PCBP2 also modulates p53 translation through binding to the 5′ terminus of p53 mRNA. Thus PCBP2 emerges as a double-function factor in the p53 expression.

Translation of human Δ133p53 mRNA and its targeting by antisense oligonucleotides complementary to the 5'-terminal region of this mRNA.

The p53 protein is expressed as at least twelve protein isoforms. Within intron 4 of the human TP53 gene, a P2 transcription initiation site is located and this transcript encodes two p53 isoforms: Δ133p53 and Δ160p53. Here, the secondary structure of the 5′-terminal region of P2-initiated mRNA was characterized by means of the SHAPE and Pb2+-induced cleavage methods and for the first time, a secondary structure model of this region was proposed. Surprisingly, only Δ133p53 isoform was synthetized in vitro from the P2-initiated p53 mRNA while translation from both initiation codons occurred after the transfection of vector-encoded model mRNA to HCT116 cells. Interestingly, translation performed in the presence of the cap analogue suggested that the cap-independent process contributes to the translation of P2-initiated p53 mRNA. Subsequently, several antisense oligonucleotides targeting the 5′-terminal region of P2-initiated p53 mRNA were designed. The selected oligomers were applied in in vitro translation assays as well as in cell lines and their impact on the Δ133p53 synthesis and on cell viability was investigated. The results show that these oligomers are attractive tools in the modulation of the translation of P2-initiated p53 mRNA through attacking the 5′ terminus of the transcript. Since cell proliferation is also reduced by antisense oligomers that lower the level of Δ133p53, this demonstrates an involvement of this isoform in tumorigenesis.

Abstract 2160: Tumor suppressor p53 down-regulates programmed cell death protein 4 (PDCD4) expression

Abstract Programmed Cell Death Protein 4 (PDCD4), a well-known tumor suppressor, acts to inhibit translation initiation and cap-dependent translation by inhibiting the helicase activity of EIF4A. EIF4A tends to target mRNAs with structured 5'-UTR. PDCD4 can also prevent tumorigenesis by inhibiting tumor promoter-induced neoplastic transformation, and studies indicate that PDCD4 may inhibit translation of certain mRNAs via directly binding. A previous study has demonstrated that PDCD4 inhibits translation of p53 mRNA and treatment with DNA-damaging agents down-regulates PDCD4 expression but activates p53 expression. The study further demonstrated that treatment with DNA-damaging agents resulted in down-regulation of PDCD4 expression and an increase in p53 expression, suggesting a potential mechanism in which p53 regulates expression of Pdcd4. However, whether p53 directly regulates PDCD4 remains unknown. Herein, we demonstrate for the first time that p53 regulates PDCD4 expression. First, we found that overexpression of p53 in p53-null cells (H1299 and Saos2 cells) decreased PDCD4 protein level. Secondly, p53 dose-dependently decreased PDCD4 promoter activity in gene reporter assays. Moreover, we demonstrated that mutations on p53 (R273H: contact mutant, hotspot and R175H: conformational mutant, hotspot) abolished p53-mediated PDCD4 repression. Furthermore, using Tet-on expression system, PDCD4 protein level was decreased when WT p53, but not hotspot mutants of p53, was expressed in H1299 cells. Finally, mutations on DNA-binding domain, but not C-terminal regulatory domain, of p53 disrupted p53-mediated PDCD4 repression. Taken together, our results suggest that p53 functions as a novel regulator of PDCD4 and the relationship between of p53 and PDCD4 may be involved in tumor development and progression. Citation Format: Wei-Hsiung Yang, Andrew P. George, William H. Yang, Chiung-Min Wang, Richard H. Yang. Tumor suppressor p53 down-regulates programmed cell death protein 4 (PDCD4) expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2160.

Fine-tuning p53 activity by modulating the interaction between eukaryotic translation initiation factor eIF4E and RNA-binding protein RBM38.

p53 is critical for tumor suppression but also elicits detrimental effects when aberrantly overexpressed. Thus, multiple regulators, including RNA-binding protein RBM38, are found to tightly control p53 expression. Interestingly, RBM38 is unique in that it can either suppress or enhance p53 mRNA translation via altered interaction with eIF4E potentially mediated by serine-195 (S195) in RBM38. Thus, multiple RBM38/eIF4E knock-in (KI) cell lines were generated to investigate the significance of eIF4E-RBM38 interaction in controlling p53 activity. We showed that KI of RBM38-S195D or -Y192C enhances, whereas KI of RBM38-S195K/R/L weakens, the binding of eIF4E to p53 mRNA and subsequently p53 expression. We also showed that KI of eIF4E-D202K weakens the interaction of eIF4E with RBM38 and thereby enhances p53 expression, suggesting that D202 in eIF4E interacts with S195 in RBM38. Moreover, we generated an Rbm38 S193D KI mouse model in which human-equivalent serine-193 is substituted with aspartic acid. We showed that S193D KI enhances p53-dependent cellular senescence and that S193D KI mice have a shortened life span and are prone to spontaneous tumors, chronic inflammation, and liver steatosis. Together, we provide in vivo evidence that the RBM38-eIF4E loop can be explored to fine-tune p53 expression for therapeutic development.

Alternative Mechanisms of p53 Action During the Unfolded Protein Response.

The tumor suppressor protein p53 orchestrates cellular responses to a vast number of stresses, with DNA damage and oncogenic activation being some of the best described. The capacity of p53 to control cellular events such as cell cycle progression, DNA repair, and apoptosis, to mention some, has been mostly linked to its role as a transcription factor. However, how p53 integrates different signaling cascades to promote a particular pathway remains an open question. One way to broaden its capacity to respond to different stimuli is by the expression of isoforms that can modulate the activities of the full-length protein. One of these isoforms is p47 (p53/47, Δ40p53, p53ΔN40), an alternative translation initiation variant whose expression is specifically induced by the PERK kinase during the Unfolded Protein Response (UPR) following Endoplasmic Reticulum stress. Despite the increasing knowledge on the p53 pathway, its activity when the translation machinery is globally suppressed during the UPR remains poorly understood. Here, we focus on the expression of p47 and we propose that the alternative initiation of p53 mRNA translation offers a unique condition-dependent mechanism to differentiate p53 activity to control cell homeostasis during the UPR. We also discuss how the manipulation of these processes may influence cancer cell physiology in light of therapeutic approaches.

A novel 3'-tRNAGlu-derived fragment acts as a tumor suppressor in breast cancer by targeting nucleolin.

tRNA-derived fragments (tRFs) have been defined as a novel class of small noncoding RNAs. tRFs have been reported to be deregulated in cancer, but their biologic function remains to be fully understood. We have identified a new tRF (named tRF3E), derived from mature tRNAGlu, that is specifically expressed in healthy mammary glands but not in breast cancer (BC). Consistently, tRF3E levels significantly decrease in the blood of patients with epidermal growth factor receptor 2 (HER2)-positive BC reflecting tumor status (control > early cancer > metastatic cancer). tRF3E down-regulation was recapitulated in Δ16HER2 transgenic mice, representing a BC preclinical model. Pulldown assays, used to search for proteins capable to selectively bind tRF3E, have shown that this tRF specifically interacts with nucleolin (NCL), an RNA-binding protein overexpressed in BC and able to repress the translation of p53 mRNA. The binding properties of NCL-tRF3E complex, predicted in silico and analyzed by EMSA assays, are congruent with a competitive displacement of p53 mRNA by tRF3E, leading to an increased p53 expression and consequently to a modulation of cancer cell growth. Here, we provide evidence that tRF3E plays an important role in the pathogenesis of BC displaying tumor-suppressor functions through a NCL-mediated mechanism.-Falconi, M., Giangrossi, M., Elexpuru Zabaleta, M., Wang, J., Gambini, V., Tilio, M., Bencardino, D., Occhipinti, S., Belletti, B., Laudadio, E., Galeazzi, R., Marchini, C., Amici, A. A novel 3'-tRNAGlu-derived fragment acts as a tumor suppressor in breast cancer by targeting nucleolin.

Integrated Regulation of HuR by Translation Repression and Protein Degradation Determines Pulsatile Expression of p53 Under DNA Damage

SummaryExpression of tumor suppressor p53 is regulated at multiple levels, disruption of which often leads to cancer. We have adopted an approach combining computational systems modeling with experimental validation to elucidate the translation regulatory network that controls p53 expression post DNA damage. The RNA-binding protein HuR activates p53 mRNA translation in response to UVC-induced DNA damage in breast carcinoma cells. p53 and HuR levels show pulsatile change post UV irradiation. The computed model fitted with the observed pulse of p53 and HuR only when hypothetical regulators of synthesis and degradation of HuR were incorporated. miR-125b, a UV-responsive microRNA, was found to represses the translation of HuR mRNA. Furthermore, UV irradiation triggered proteasomal degradation of HuR mediated by an E3-ubiquitin ligase tripartite motif-containing 21 (TRIM21). The integrated action of miR-125b and TRIM21 constitutes an intricate control system that regulates pulsatile expression of HuR and p53 and determines cell viability in response to DNA damage.

Abstract 3958: Inhibiting the inhibitor: Exploring the p53-Rbm38 loop as a potential cancer therapeutic target

Abstract Modulating Rbm38 negative regulation of p53 translation may be an effective therapeutic target in human cancers. As a focal regulatory component of RNA metabolism, RNA-binding proteins (RBPs) modulate all facets of RNA biogenesis. Given the fact that RBPs control RNA metabolism, defects in the function of RBPs can have far reaching implications including neurodegenerative disorders and cancer. Our group discovered that Rbm38 suppresses p53 mRNA translation by interacting with eukaryotic translation initiation factor 4E (eIF4E) on p53 mRNA. Conceptually, blocking the Rbm38 interaction with eIF4E employing synthetic peptides corresponding to their respective binding interface should relieve the translational repression caused by Rbm38. Indeed, treating cancer cells with synthetic peptides corresponding to the binding interface between Rbm38 and eIF4E resulted in robust p53 protein expression. Moreover, addition of peptide and low dose doxorubicin caused further accumulation of p53. Consequently, treatment of cancer cells with peptide alone or in combination with low dose doxorubicin leads to a decrease in colony formation as well as tumor sphere formation. Of significance, RKO xenografts with wild-type p53 were highly sensitive to treatment with a synthetic peptide comprising just 8 amino acids (Pep8) compared to the control group. Together, these data support that p53 expression can be modulated by using small peptides to inhibit Rbm38 from interacting with eIF4E, which may be used as a therapeutic approach in cancers carrying wild-type p53. Citation Format: Chris A. Lucchesi. Inhibiting the inhibitor: Exploring the p53-Rbm38 loop as a potential cancer therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3958.

Induction of the p53 Tumor Suppressor in Cancer Cells through Inhibition of Cap-Dependent Translation.

The p53 tumor suppressor plays a critical role in protecting normal cells from malignant transformation. Development of small molecules to reactivate p53 in cancer cells has been an area of intense research. We previously identified an internal ribosomal entry site (IRES) within the 5' untranslated region of p53 mRNA that mediates translation of the p53 mRNA independent of cap-dependent translation. Our results also show that in response to DNA damage, cells switch from cap-dependent translation to cap-independent translation of p53 mRNA. In the present study, we discovered a specific inhibitor of cap-dependent translation, 4EGI-1, that is capable of inducing the accumulation of p53 in cancer cells retaining wild-type p53. Our results show that 4EGI-1 causes an increase in p53 IRES activity, leading to increased translation of p53 mRNA. We also observed that 4EGI-1 induces cancer cell apoptosis in a p53-dependent manner. Furthermore, 4EGI-1 induces p53 in cancer cells without causing DNA double-strand breaks. In conclusion, we discovered a mechanistic link between inhibition of cap-dependent translation and enhanced p53 accumulation. This leads to apoptosis of cancer cells without causing collateral damage to normal cells, thus providing a novel and effective therapeutic strategy for cancer.

Rbm24, a target of p53, is necessary for proper expression of p53 and heart development.

Activation of p53-dependent apoptosis is critical for tumor suppression but aberrant activation of p53 also leads to developmental defects and heart failure. Here, we found that Rbm24 RNA-binding protein, a target of p53, regulates p53 mRNA translation. Mechanistically, we found that through binding to p53 mRNA and interaction with translation initiation factor eIF4E, Rbm24 prevents eIF4E from binding to p53 mRNA and inhibits the assembly of translation initiation complex. Importantly, we showed that mice deficient in Rbm24 die in utero due to the endocardial cushion defect in the heart at least in part due to aberrant activation of p53-dependent apoptosis. We also showed that the heart developmental defect in Rbm24-null mice can be partially rescued by p53 deficiency through decreased apoptosis in the heart. Together, we postulate that the p53-Rbm24 loop is critical for the heart development and may be explored for mitigating congenital heart diseases and heart failure.

Ninjurin 1 has two opposing functions in tumorigenesis in a p53-dependent manner

WT p53 is critical for tumor suppression, whereas mutant p53 promotes tumor progression. Nerve injury-induced protein 1 (Ninj1) is a target of p53 and forms a feedback loop with p53 by repressing p53 mRNA translation. Here, we show that loss of Ninj1 increased mutant p53 expression and, subsequently, enhanced cell growth and migration in cells carrying a mutant p53. In contrast, loss of Ninj1 inhibited cell growth and migration in cells carrying a WT p53. To explore the biological significance of Ninj1, we generated a cohort of Ninj1-deficient mice and found that Ninj1+/- mice were prone to systemic inflammation and insulitis, but not to spontaneous tumors. We also found that loss of Ninj1 altered the tumor susceptibility in both mutant p53 and p53-null background. Specifically, in a mutant p53(R270H) background, Ninj1 deficiency shortened the lifespan, altered the tumor spectrum, and increased tumor burden, likely via enhanced expression of mutant p53. In a p53-null background, Ninj1 deficiency significantly increased the incidence of T-lymphoblastic lymphoma. Taken together, our data suggest that depending on p53 genetic status, Ninj1 has two opposing functions in tumorigenesis and that the Ninj1-p53 loop may be targeted to manage inflammatory diseases and cancer.

DDX3 localizes to the centrosome and prevents multipolar mitosis by epigenetically and translationally modulating p53 expression

The DEAD-box RNA helicase DDX3 plays divergent roles in tumorigenesis, however, its function in mitosis is unclear. Immunofluorescence indicated that DDX3 localized to centrosome throughout the cell cycle and colocalized with centrosome-associated p53 during mitosis in HCT116 and U2OS cells. DDX3 depletion promoted chromosome misalignment, segregation defects and multipolar mitosis, eventually leading to G2/M delay and cell death. DDX3 prevented multipolar mitosis by inactivation and coalescence of supernumerary centrosomes. DDX3 silencing suppressed Ser15 phosphorylation of p53 which is required for p53 centrosomal localization. Additionally, knockout of p53 dramatically diminished the association of DDX3 with centrosome, which was rescued by overexpression of the centrosomal targeting-defective p53 S15A mutant, indicating that centrosomal localization of DDX3 is p53 dependent but not through centrosomal location of p53. Furthermore, DDX3 knockdown suppressed p53 transcription through activation of DNA methyltransferases (DNMTs) along with hypermethylation of p53 promoter and promoting the binding of repressive histone marks to p53 promoter. Moreover, DDX3 modulated p53 mRNA translation. Taken together, our study suggests that DDX3 regulates epigenetic transcriptional and translational activation of p53 and colocalizes with p53 at centrosome during mitosis to ensure proper mitotic progression and genome stability, which supports the tumor-suppressive role of DDX3.

Ferredoxin reductase is critical for p53-dependent tumor suppression via iron regulatory protein 2.

Ferredoxin reductase (FDXR), a target of p53, modulates p53-dependent apoptosis and is necessary for steroidogenesis and biogenesis of iron-sulfur clusters. To determine the biological function of FDXR, we generated a Fdxr-deficient mouse model and found that loss of Fdxr led to embryonic lethality potentially due to iron overload in developing embryos. Interestingly, mice heterozygous in Fdxr had a short life span and were prone to spontaneous tumors and liver abnormalities, including steatosis, hepatitis, and hepatocellular carcinoma. We also found that FDXR was necessary for mitochondrial iron homeostasis and proper expression of several master regulators of iron metabolism, including iron regulatory protein 2 (IRP2). Surprisingly, we found that p53 mRNA translation was suppressed by FDXR deficiency via IRP2. Moreover, we found that the signal from FDXR to iron homeostasis and the p53 pathway was transduced by ferredoxin 2, a substrate of FDXR. Finally, we found that p53 played a role in iron homeostasis and was required for FDXR-mediated iron metabolism. Together, we conclude that FDXR and p53 are mutually regulated and that the FDXR-p53 loop is critical for tumor suppression via iron homeostasis.

Heterogeneous nuclear ribonucleoprotein (hnRNP) L promotes DNA damage-induced cell apoptosis by enhancing the translation of p53.

The tumor suppressor p53 is an essential gene in the induction of cell cycle arrest, DNA repair, and apoptosis. p53 protein is induced under cellular stress, blocking cell cycle progression and inducing DNA repair. Under DNA damage conditions, it has been reported that post-transcriptional regulation of p53 mRNA contributes to the increase in p53 protein level. Here we demonstrate that heterogeneous nuclear ribonucleoprotein (hnRNP) L enhances p53 mRNA translation. We found that hnRNP L is increased and binds to the 5’UTR of p53 mRNA in response to DNA damage. Increased hnRNP L caused enhancement of p53 mRNA translation. Conversely, p53 protein levels were decreased following hnRNP L knock-down, rendering them resistant to apoptosis and arrest in the G2/M phase after DNA damage. Thus, our findings suggest that hnRNP L functions as a positive regulator of p53 translation and promotes cell cycle arrest and apoptosis.

Interplay between RNA-binding protein HuR and microRNA-125b regulates p53 mRNA translation in response to genotoxic stress

ABSTRACTTumor suppressor protein p53 plays a crucial role in maintaining genomic integrity in response to DNA damage. Regulation of translation of p53 mRNA is a major mode of regulation of p53 expression under genotoxic stress. The AU/U-rich element-binding protein HuR has been shown to bind to p53 mRNA 3′UTR and enhance translation in response to DNA-damaging UVC radiation. On the other hand, the microRNA miR-125b is reported to repress p53 expression and stress-induced apoptosis. Here, we show that UVC radiation causes an increase in miR-125b level in a biphasic manner, as well as nuclear cytoplasmic translocation of HuR. Binding of HuR to the p53 mRNA 3′UTR, especially at a site adjacent to the miR-125b target site, causes dissociation of the p53 mRNA from the RNA-induced silencing complex (RISC) and inhibits the miR-125b-mediated translation repression of p53. HuR prevents the oncogenic effect of miR-125b by reversing the decrease in apoptosis and increase in cell proliferation caused by the overexpression of miR-125b. The antagonistic interplay between miR-125b and HuR might play an important role in fine-tuning p53 gene expression at the post-transcriptional level, and thereby regulate the cellular response to genotoxic stress.

A novel cytoprotective function for the DNA repair protein Ku in regulating p53 mRNA translation andfunction.

Ku heterodimer is a DNA binding protein with a prominent role in DNA repair. Here, we investigate whether and how Ku impacts the DNA damage response by acting as a post-transcriptional regulator of gene expression. We show that Ku represses p53 protein synthesis and p53-mediated apoptosis by binding to a bulged stem-loop structure within the p53 5' UTR However, Ku-mediated translational repression of the p53 mRNA is relieved after genotoxic stress. The underlying mechanism involves Ku acetylation which disrupts Ku-p53 mRNA interactions. These results suggest that Ku-mediated repression of p53 mRNA translation constitutes a novel mechanism linking DNA repair and mRNA translation.

Translation, Pdcd4 and eIF4A.

Pdcd4 (programmed cell death 4) has received considerable attention as a tumor suppressor protein in recent years, however, its molecular function is still poorly understood. Pdcd4 is a nuclear-cytoplasmic shuttling and RNA-binding protein, which is involved in the control of translation of specific mRNAs. Pdcd4 interacts with the eukaryotic translation initiation factor eIF4A, an RNA helicase that plays a critical role in cap-dependent translation by melting stable RNA secondary structures in the 5′-untranslated regions (UTRs) of mRNAs [1,2]. It has been shown that Pdcd4 inhibits the helicase activity of eIF4A, suggesting that it suppresses translation of mRNAs with highly structured 5′-UTRs [3]. This idea was supported by analyzing the effect of Pdcd4 on artificial RNA constructs containing stable hairpin structures in the 5′-UTR and, more recently, confirmed by demonstrating that translation of p53 mRNA (whose 5′-UTR forms very stable secondary structures) is suppressed by Pdcd4 via an eIF4A-dependent mechanism [4] (Figure ​(Figure1a1a). Figure 1 Suppression of translation initiation (a) and translation elongation (b) by Pdcd4 Recent findings indicate that the role of Pdcd4 in translation is more complex and involves an additional, entirely different inhibitory mechanism. It was shown that Pdcd4 suppresses the translation of the c-myb and A-myb mRNAs even when the eIF4A binding site was destroyed by mutation [5,6]. Instead, the RNA-binding activity of Pdcd4 was required to suppress translation of these RNAs, suggesting that direct RNA-binding by Pdcd4 plays a key role. Additional work revealed that the nucleotide sequences responsible for Pdcd4-induced translation suppression were located in the coding regions of c-myb and A-myb mRNAs. Furthermore, in vitro RNA-binding studies demonstrated that the “Pdcd4 response regions” of the c-myb and A-myb mRNAs are able to form secondary structures which were preferentially bound by Pdcd4. Overall, these experiments suggested that Pdcd4 is able to suppress translation by a novel mechanism, which involves direct binding of Pdcd4 to specific target RNAs. How does Pdcd4 suppress translation from within the coding region? When the c-myb coding region was placed under the control of the Hepatitis C virus internal ribosomal entry site (HCV-IRES) Pdcd4 failed to suppress translation [5]. Because the HCV-IRES does not depend on the translation initiation factors required for cap-dependent initiation, this suggested that Pdcd4 suppresses translation of c-myb mRNA by interfering with one of these factors (except eIF4A) at translation initiation. However, when the c-myb and A-myb “Pdcd4 response regions” were fused to GFP RNA to ask if they are able to convey Pdcd4-responsiveness onto a heterologous RNA, a surprising observation was made. It was indeed found that Pdcd4 was able to suppress translation of the recombinant RNA, but only when a continuous open reading frame extended from the GFP coding sequence into the added c-myb or A-myb sequences. In other words, Pdcd4 suppressed the translation of the recombinant RNAs only when the binding region for Pdcd4 was itself part of the open reading frame. This observation was confirmed by introducing in-frame translational stop codons into the authentic c-myb and A-myb coding regions upstream of the “Pdcd4 response regions”. This completely abolished the inhibitory effect of Pdcd4, again indicating that Pdcd4 supresses translation only when the sequence to which it binds is part of the translated region [6]. Thus, truncating the coding region by a single stop codon is sufficient to abrogate Pdcd4-dependent inhibition. A straightforward explanation for this observation is that Pdcd4 suppresses translation of these RNAs at the elongation step (Figure ​(Figure1b1b). How can this be reconciled with the fact that the translation of the c-myb coding region was not suppressed by Pdcd4 when translation was initiated at the HCV-IRES? A possible explanation comes from the observation that the efficiency of IRES-dependent translation initiation was much lower than cap-dependent translation [5]. If the inhibition of translation elongation by Pdcd4 is augmented with increasing translation efficiency, it suggests that Pdcd4 acts like a self-adjusting controller, that limits the translation output when the translation rate is high but has no effect when it is low. How Pdcd4 actually suppresses translation at elongation is currently unknown. Binding of Pdcd4 could stabilize RNA secondary structures and thereby hinder the passage of approaching ribosomes. Pdcd4 also interacts with the poly(A) binding protein which, in turn, could stabilize the binding of Pdcd4 to the response region [7]. Elongating ribosomes might also be blocked in an active manner. Such a mechanism has been described for the cytoplasmic polyadenylation element binding protein CPEB2, which interacts with the elongation factor eEF2 and reduces eEF2/ribosome-triggered GTP hydrolysis, thereby slowing down translation elongation of CPEB2-bound RNAs [8]. In any case, the work discussed here has led to a new paradigm for translational suppression and recognition of specific target RNAs by Pdcd4. Exploring its relevance to the function of Pdcd4 as a tumor suppressor will now be an important task.