A Novel High Throughput Biochemical Assay to Evaluate the HuR Protein-RNA Complex Formation

D’Agostino, Vito; Adami, Valentina; Provenzani, Alessandro

doi:10.1371/journal.pone.0072426

Cited by 61 publications

(84 citation statements)

References 35 publications

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“…27 This assay includes four components: His6 -tagged HuR RRM1/2 domain, biotinylated ARE Msi1 oligo, streptavidin coated donor beads, and nickel coated acceptor beads. The interaction of RRM1/2 and ARE Msi1 brings the two beads close enough to generate a signal following excitation.…”

Section: Resultsmentioning

confidence: 99%

Identification and Validation of Novel Small Molecule Disruptors of HuR-mRNA Interaction

et al. 2015

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HuR, an RNA binding protein, binds to adenine- and uridine-rich elements (ARE) in the 3′-untranslated region (UTR) of target mRNAs, regulating their stability and translation. HuR is highly abundant in many types of cancer, and it promotes tumorigenesis by interacting with cancer-associated mRNAs, which encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. Drugs that disrupt the stabilizing effect of HuR upon mRNA targets could have dramatic effects on inhibiting cancer growth and persistence. In order to identify small molecules that directly disrupt the HuR–ARE interaction, we established a fluorescence polarization (FP) assay optimized for high throughput screening (HTS) using HuR protein and an ARE oligo from Musashi RNA-binding protein 1 (Msi1) mRNA, a HuR target. Following the performance of an HTS of ~6000 compounds, we discovered a cluster of potential disruptors, which were then validated by AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay), surface plasmon resonance (SPR), ribonucleoprotein immunoprecipitation (RNP IP) assay, and luciferase reporter functional studies. These compounds disrupted HuR–ARE interactions at the nanomolar level and blocked HuR function by competitive binding to HuR. These results support future studies toward chemical probes for a HuR function study and possibly a novel therapy for HuR-overexpressing cancers.

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Section: Resultsmentioning

confidence: 99%

Identification and Validation of Novel Small Molecule Disruptors of HuR-mRNA Interaction

et al. 2015

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“…Next, to quantify the binding affinity of RdRp with 3Ј-SL, we exploited an Alpha assay with 5Ј-biotinylated RNA substrates and N-terminally His-tagged RdRp. The Alpha technology has recently been used to determine interactions of RNA-binding proteins in solution (19). We employed truncated forms of 5Ј-SLA and 3Ј-SL RNAs in the binding assay to narrow down the interaction site (Fig.…”

Section: Isolation Of Viral Rna Elements That Interact With Rdrp Viamentioning

confidence: 99%

Identification of a Conserved RNA-dependent RNA Polymerase (RdRp)-RNA Interface Required for Flaviviral Replication

Hodge

Tunghirun

Kamkaew

et al. 2016

Journal of Biological Chemistry

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Dengue virus, an ϳ10.7-kb positive-sense RNA virus, is the most common arthropod-communicated pathogen in the world. Despite dengue's clear epidemiological importance, mechanisms for its replication remain elusive. Here, we probed the entire dengue genome for interactions with viral RNA-dependent RNA polymerase (RdRp), and we identified the dominant interaction as a loop-forming ACAG motif in the 3 positive-stranded terminus, complicating the prevailing model of replication. A subset of interactions coincides with known flaviviral recombination sites inside the viral protein-coding region. Specific recognition of the RNA element occurs via an arginine patch in the C-terminal thumb domain of RdRp. We also show that the highly conserved nature of the consensus RNA motif may relate to its tolerance to various mutations in the interacting region of RdRp. Disruption of the interaction resulted in loss of viral replication ability in cells. This unique RdRp-RNA interface is found throughout flaviviruses, implying possibilities for broad disease interventions.Positive-sense single-stranded RNA viruses initiate replication by the generation of a complementary negative (Ϫ) RNA strand via the action of viral RNA-dependent RNA polymerases (RdRps). 3 In the cases of brome mosaic virus (family Bromoviridae), turnip crinkle virus (Tombusviridae), hepatitis C virus (Flaviviridae), and encephalomyocarditis virus (Picornaviridae), RNA structures in the 3Ј-untranslated region (UTR) of the positive strand have been characterized as RdRp promoters (1-4). In the case of dengue virus (DENV), a flavivirus with a 5Ј-type I cap and the absence of a polyadenylated tail, however, the current model for replication asserts that the 5Ј-UTR acts as a promoter for (Ϫ)-strand synthesis. This position is supported by several lines of in vitro evidence. In particular, atomic force microscopy demonstrated cyclization of the (ϩ)-strand genomic RNA, resulting in placement of the 5Ј-UTR in proximity to the 3Ј terminus, whereas EMSA and footprinting assays apparently documented RdRp interactions with the first hairpin element in the DENV 5Ј-UTR, designated stem-loop A (SLA) (5). In addition, an RNA fragment of the DENV 5Ј-UTR could also stimulate in vitro synthesis of a complementary product of the 3Ј-UTR (5, 6).To date, investigations of RdRp actions on the flaviviral RNA genome have been limited to in vitro assays on less than 5% of the whole genome with a bias toward UTR regions; therefore, the entire interaction landscape of RdRp has not yet been revealed. It is also unclear whether flaviviral RdRps require a specific RNA promoter for de novo initiation of RNA synthesis. To gain insight into accurate mechanisms for synthesis of the viral genome in the cell, here we explored all possible interactions between RdRp and the complete RNA genome in an unbiased in vivo context using a refined yeast three-hybrid (Y3H) scan. Combining bioinformatic evidence with in vitro binding and viral replicon assays, we pinpoint amino acids and nucleotides contributin...

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“…Our research indicates that through pharmacological inhibition of HuR RNA binding, by means of MTX (D'Agostino et al, 2013) or HuR genetic ablation, SOX2 mRNA is stabilized, accompanied by an increase in protein expression. These results support the strong role of HuR in SOX2 mRNA processing and turnover.…”

Section: Discussionmentioning

confidence: 75%

Human Antigen R Binding and Regulation of SOX2 mRNA in Human Mesenchymal Stem Cells

et al. 2015

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Since 2005, sex determining region y-box 2 (SOX2) has drawn the attention of the scientific community for being one of the key transcription factors responsible for pluripotency induction in somatic stem cells. Our research investigated the turnover regulation of SOX2 mRNA in human adipose-derived stem cells, considered one of the most valuable sources of somatic stem cells in regenerative medicine. Mitoxantrone is a drug that acts on nucleic acids primarily used to treat certain types of cancer and was recently shown to ameliorate the outcome of autoimmune diseases such as multiple sclerosis. In addition, mitoxantrone has been shown to inhibit the binding of human antigen R (HuR) RNA-binding protein to tumor necrosis factor-a mRNA. Our results show that HuR binds to the 39-untranslated region of SOX2 mRNA together with the RNAinduced silencing complex miR145. The HuR binding works by stabilizing the interaction between the 39-untranslated region and the RNA-induced silencing complex. Cell exposure to mitoxantrone leads to HuR detachment and the subsequent prolongation of the SOX2 mRNA half-life. The prolonged SOX2 half-life allows improvement of the spheroid-forming capability of the adipose-derived stem cells. The silencing of HuR confirmed the above observations and illustrates how the RNA-binding protein HuR may be a required molecule for regulation of SOX2 mRNA decay.

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A Novel High Throughput Biochemical Assay to Evaluate the HuR Protein-RNA Complex Formation

Cited by 61 publications

References 35 publications

Identification and Validation of Novel Small Molecule Disruptors of HuR-mRNA Interaction

Identification and Validation of Novel Small Molecule Disruptors of HuR-mRNA Interaction

Identification of a Conserved RNA-dependent RNA Polymerase (RdRp)-RNA Interface Required for Flaviviral Replication

Human Antigen R Binding and Regulation of SOX2 mRNA in Human Mesenchymal Stem Cells

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