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Key Points Post-transcriptional regulators of gene regulation, such as RNA-binding proteins (RBPs) and microRNAs (miRNAs), often have many thousands of binding sites in the transcriptome. Transcription renders the concentrations of these binding sites highly dynamic. To understand their function, we consider how binding site occupancy is determined by the composition of the transcriptome and by the regulator concentration in a simple steady-state model. Competition between many binding sites prevents saturation. This buffering helps to explain how changes in the expression of an RBP or a miRNA can regulate targets with different affinities inside a cell. Expression of many strong binding sites can reduce occupancies by sequestration (that is, the 'sponge' effect). However, the larger the number of competing binding sites, the weaker the crosstalk between individual transcripts. A sponge requires approximately double the number of binding sites in order to be effective. For miRNAs this requires tens of thousands of additional binding sites, which is unlikely for typical mRNAs. Crosstalk between mRNAs could be enhanced if local concentrations deviate strongly from the average or if multiple sites cooperate. Increasing evidence suggests that competition between transcripts for binding of microRNAs and RNA-binding proteins might be a fundamental principle of post-transcriptional gene regulation. The authors use a simple steady-state model to quantitatively assess competition effects under physiological conditions and review the role of endogenous 'sponges' in light of the key features that emerge. Post-transcriptional gene regulation (PTGR) of mRNA turnover, localization and translation is mediated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). These regulators exert their effects by binding to specific sequences within their target mRNAs. Increasing evidence suggests that competition for binding is a fundamental principle of PTGR. Not only can miRNAs be sequestered and neutralized by the targets with which they interact through a process termed 'sponging', but competition between binding sites on different RNAs may also lead to regulatory crosstalk between transcripts. Here, we quantitatively model competition effects under physiological conditions and review the role of endogenous sponges for PTGR in light of the key features that emerge.

Serotonin (5-HT) and oxytocin (OXT) are two neuromodulators involved in human affect and sociality and in disorders like depression and autism. We asked whether these chemical messengers interact in the regulation of emotion-based behavior by administering OXT or placebo to 24 healthy subjects and mapping cerebral 5-HT system by using 2′-methoxyphenyl-(N-2′-pyridinyl)-p-[ ¹⁸F]fluoro-benzamidoethylpiperazine ([ ¹⁸F]MPPF), an antagonist of 5-HT ₁A receptors. OXT increased [ ¹⁸F]MPPF nondisplaceable binding potential (BP ND) in the dorsal raphe nucleus (DRN), the core area of 5-HT synthesis, and in the amygdala/hippocampal complex, insula, and orbitofrontal cortex. Importantly, the amygdala appears central in the regulation of 5-HT by OXT: [ ¹⁸F]MPPF BP ND changes in the DRN correlated with changes in right amygdala, which were in turn correlated with changes in hippocampus, insula, subgenual, and orbitofrontal cortex, a circuit implicated in the control of stress, mood, and social behaviors. OXT administration is known to inhibit amygdala activity and results in a decrease of anxiety, whereas high amygdala activity and 5-HT dysregulation have been associated with increased anxiety. The present study reveals a previously unidentified form of interaction between these two systems in the human brain, i.e., the role of OXT in the inhibitory regulation of 5-HT signaling, which could lead to novel therapeutic strategies for mental disorders.

Impaired turnover of the autophagy substrate p62 leads to liver injury. p62 inhibits the ubiquitin ligase Keap1, leading to stabilization of the transcription factor Nrf2. High levels of p62 in autophagy deficient animals leads to unusually high expression of Nrf2 targets genes and results in liver injury. Impaired selective turnover of p62 by autophagy causes severe liver injury accompanied by the formation of p62-positive inclusions and upregulation of detoxifying enzymes. These phenotypes correspond closely to the pathological conditions seen in human liver diseases, including alcoholic hepatitis and hepatocellular carcinoma. However, the molecular mechanisms and pathophysiological processes in these events are still unknown. Here we report the identification of a novel regulatory mechanism by p62 of the transcription factor Nrf2, whose target genes include antioxidant proteins and detoxification enzymes. p62 interacts with the Nrf2-binding site on Keap1, a component of Cullin-3-type ubiquitin ligase for Nrf2. Thus, an overproduction of p62 or a deficiency in autophagy competes with the interaction between Nrf2 and Keap1, resulting in stabilization of Nrf2 and transcriptional activation of Nrf2 target genes. Our findings indicate that the pathological process associated with p62 accumulation results in hyperactivation of Nrf2 and delineates unexpected roles of selective autophagy in controlling the transcription of cellular defence enzyme genes.

Peptide binding to major histocompatibility complexes (MHCs) is a central component of the immune system, and understanding the mechanism behind stable peptide–MHC binding will aid the development of immunotherapies. While MHC binding is mostly influenced by the identity of the so-called anchor positions of the peptide, secondary interactions from nonanchor positions are known to play a role in complex stability. However, current MHC-binding prediction methods lack an analysis of the major conformational states and might underestimate the impact of secondary interactions. In this work, we present an atomically detailed analysis of peptide–MHC binding that can reveal the contributions of any interaction toward stability. We propose a simulation framework that uses both umbrella sampling and adaptive sampling to generate a Markov state model (MSM) for a coronavirus-derived peptide (QFKDNVILL), bound to one of the most prevalent MHC receptors in humans (HLA-A24:02). While our model reaffirms the importance of the anchor positions of the peptide in establishing stable interactions, our model also reveals the underestimated importance of position 4 (p4), a nonanchor position. We confirmed our results by simulating the impact of specific peptide mutations and validated these predictions through competitive binding assays. By comparing the MSM of the wild-type system with those of the D4A and D4P mutations, our modeling reveals stark differences in unbinding pathways. The analysis presented here can be applied to any peptide–MHC complex of interest with a structural model as input, representing an important step toward comprehensive modeling of the MHC class I pathway.

The pluripotency transcription factor SOX2 is essential for the maintenance of glioblastoma stem cells (GSC), which are thought to underlie tumor growth, treatment resistance, and recurrence. To understand how SOX2 is regulated in GSCs, we utilized a proteomic approach and identified the E3 ubiquitin ligase TRIM26 as a direct SOX2-interacting protein. Unexpectedly, we found TRIM26 depletion decreased SOX2 protein levels and increased SOX2 polyubiquitination in patient-derived GSCs, suggesting TRIM26 promotes SOX2 protein stability. Accordingly, TRIM26 knockdown disrupted the SOX2 gene network and inhibited both self-renewal capacity as well as in vivo tumorigenicity in multiple GSC lines. Mechanistically, we found TRIM26, via its C-terminal PRYSPRY domain, but independent of its RING domain, stabilizes SOX2 protein by directly inhibiting the interaction of SOX2 with WWP2, which we identify as a bona fide SOX2 E3 ligase in GSCs. Our work identifies E3 ligase competition as a critical mechanism of SOX2 regulation, with functional consequences for GSC identity and maintenance. SOX2 is required for the maintenance of glioblastoma stem cells (GSCs). Here the authors identify that the RING family E3 ubiquitin ligase TRIM26 promotes SOX2 stability in a non-canonical ligase-independent manner and thus, increases the tumorigenicity of GSCs.

It has been found that long noncoding RNA HOTAIR, microRNA‐130a (miR‐130a) and insulin‐like growth factor 1 (IGF1) expression are associated with ovarian cancer, thus, we hypothesised that the HOTAIR/miR‐130a/IGF1 axis might associate with endocrine disorders and biological behaviours of ovarian granulosa cells in rat models of polycystic ovary syndrome (PCOS). PCOS rat models were established by injection of dehydro‐isoandrosterone, followed by treatment of si‐HOTAIR, oe‐HOTAIR, miR‐130a mimics or miR‐130a inhibitors. Serum hormonal levels were determined to evaluate endocrine conditions. The effect of HOTAIR and miR‐130a on activities of isolated ovarian granulosa cells was assessed, as well as the involvement of IGF1.In the ovarian tissues and granulosa cells of PCOS rat models, highly expressed HOTAIR and IGF1 and poorly expressed miR‐130a were identified. In response to oe‐HOTAIR, serum levels of E2, T and LH were increased and serum levels of FSH were reduced; the proliferation of granulosa cells was reduced and apoptosis was promoted; notably, expression of miR‐130a was reduced while expression of IGF1 was increased. The treatment of si‐HOTAIR reversed the situation. Furthermore, the binding of HOTAIR to miR‐130a and targeting relationship of miR‐130a and IGF1 were confirmed. LncRNA HOTAIR up‐regulates the expression of IGF1 and aggravates the endocrine disorders and granulosa cell apoptosis through competitive binding to miR‐130a in rat models of PCOS. Based on our finding, we predict that competitive binding of HOTAIR to miR‐130a may act as a novel target for the molecular treatment of PCOS.

Induction of bone formation by Wnt ligands is inhibited when sclerostin (Scl), an osteocyte-produced antagonist, binds to its receptors, the low-density lipoprotein receptor-related proteins 5 or 6 (LRP5/6). Recently, it was shown that enhanced inhibition is achieved by Scl binding to the co-receptor LRP4. However, it is not clear if the binding of Scl to LRP4 facilitates Scl binding to LRP5/6 or inhibits the Wnt pathway in an LRP5/6-independent manner. Here, using the yeast display system, we demonstrate that Scl exhibits a stronger binding affinity for LRP4 than for LRP6. Moreover, we found stronger Scl binding to LRP6 in the presence of LRP4. We further show that a Scl mutant (Scl N93A ), which tightly binds LRP4 but not LRP6, does not inhibit the Wnt pathway on its own. We demonstrate that Scl N93A competes with Scl for a common binding site on LRP4 and antagonizes Scl inhibition of the Wnt signaling pathway in osteoblasts in vitro. Finally, we demonstrate that 2 weeks of bi-weekly subcutaneous injections of Scl N93A fused to the fragment crystallizable (Fc) domain of immunoglobulin (Scl N93A Fc), which retains the antagonistic activity of the mutant, significantly increases bone formation rate and enhances trabecular volumetric bone fraction, trabecular number, and bone length in developing mice. Our data show that LRP4 serves as an anchor that facilitates Scl–LRP6 binding and that inhibition of the Wnt pathway by Scl depends on its prior binding to LRP4. We further provide evidence that compounds that inhibit Scl–LRP4 interactions offer a potential strategy to promote anabolic bone functions.

Dengue virus, an arbovirus, leads to millions of infections every year ultimately leading to a high rate of mortality. Highly effective and specific therapeutic option is not available till date to combat viral infection. One of the first stages in the virus lifecycle encompasses the viral entry into the host cell which is mediated by the interaction between heparan sulphate and the Dengue virus envelope protein in turn leading to the interaction between the envelope protein receptor binding domain and host cell receptors. The heparan sulphate binding site on the envelope protein was established using literature survey and the result validated using ColDock simulations. We have performed virtual screening against the heparan sulphate binding site using DrugBank database and short-listed probable inhibitors based on binding energy calculation following Molecular Dynamics (MD) simulations in this study. Two compounds (PubChem IDS 448062 and 656615) were selected for further analyses on which RAMD simulations were performed to quantitate the binding stability of both the molecules in the protein binding pocket which ultimately led to the selection of ZK-806450 molecule as the final selected compound. Competitive binding MD simulation against dengue virus envelope protein was performed for this molecule and heparan sulphate in order to ascertain the efficiency of binding of this molecule to the dengue virus envelope protein in the presence of its natural ligand molecule and found that this molecule has a higher affinity for the dengue virus envelope protein GAG binding site than heparan sulphate. This study may help in the use of this inhibitor molecule to combat dengue virus infection in foreseeable future and open a new avenue for drug repurposing methodology using competitive binding MD simulation.

BACKGROUND: Various lines of evidence have shown that bisphenol A [BPA;$HO-C_{6}H_{4};-C(CH_{3})_{2}- C_{6}H&_{4}-OH$] acts as an endocrine disruptor when present in very low doses. We have recently demonstrated that BPA binds strongly to human estrogen-related$receptor-\\gamma$($ERR-\\gamma$) in a binding assay using$[^{3}H]4-hydroxytamoxifen$($[^{3}H]4-OHT$). We also demonstrated that BPA inhibits the deactivation activity of 4-OHT. OBJECTICES: In the present study, we intended to obtain direct evidence that BPA interacts with$ERR-\\gamma$as a strong binder, and also to clarify the structural requirements of BPA for its binding to$ERR-\\gamma$. METHODS: We examined$[^{3}H]BPA$in the saturation binding assay using the ligand binding domain of$ERR-\\gamma$and analyzed the result using Scatchard plot analysis. A number of BPA derivatives were tested in the competitive binding assay using$[^{3}H]BPA$as a tracer and in the luciferase reporter gene assay. RESULTS:$[^{3}H]BPA$showed a KDof 5.50 nM at a Bmaxof 14.4 nmol/mg. When we examined BPA derivatives to evaluate the structural essentials required for the binding of BPA to$ERR-\\gamma$, we found that only one of the two phenol-hydroxyl groups was essential for the full binding. The maximal activity was attained when one of the methyl groups was removed. All of the potent BPA derivatives retained a high constitutive basal activity of$ERR-\\gamma$in the luciferase reporter gene assay and exhibited a distinct inhibitory activity against 4-OHT. CONCLUSION: These results indicate that the phenol derivatives are potent candidates for the endocrine disruptor that binds to$ERR-\\gamma$.

MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP) assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies.