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Lipopolysaccharide exposure to macrophages induces an inflammatory response that is heavily regulated at the transcriptional and post-transcriptional levels. HuR (ELAVL1) is an RNA binding protein that binds and regulates the maturation and half-life of AU/U rich elements (ARE) containing cytokines and chemokines transcripts, mediating the LPS-induced response. Here we investigated how and to what extent small molecule tanshinone mimics (TMs) inhibiting HuR-RNA interaction counteract LPS stimulus in macrophages. We show TMs exist in solution in keto-enolic tautomerism and that, by molecular dynamic calculations, the orto quinone form is the bioactive species interacting with HuR and inhibiting its binding mode vs mRNA targets. A chemical blockage of the diphenolic, reduced form as a diacetate caused the loss of activity of TMsin vitrobut resulted to prodrug-like activityin vivo. The murine macrophage cell line RAW264.7 was treated with LPS and TMs, and the modulation of cellular LPS-induced response was monitored by RNA and Ribonucleoprotein immunoprecipitation sequencing. Correlation analyses indicated that LPS induced a strong coupling between differentially expressed genes and HuR-bound genes, and that TMs reduced such interactions. Functional annotation addressed a specific set of genes involved in chemotaxis and immune response, such asCxcl10, Il1b, Cd40, andFas, with a decreased association with HuR, a reduction of their expression and protein secretion. The same effect was observed in primary murine bone marrow-derived macrophages, andin vivoin an LPS induced peritonitis model, in which the serum level of Cxcl10 and Il1b was strongly reduced, endowing TMs such asTM7noxwith remarkable anti-inflammatory propertiesin vivo.

YTHDF proteins bind the

Matrin3 (MATR3) is a nuclear RNA/DNA binding protein that plays pleiotropic roles in gene expression regulation by directly stabilizing target RNAs and supporting the activity of transcription factors by modulating chromatin architecture. MATR3 is involved in the differentiation of neural cells and, here, we elucidate its critical functions in regulating pluripotent circuits in human induced Pluripotent Stem Cells (hiPSCs). MATR3 downregulation affects hiPSCs differentiation potential by altering the expression level of key pluripotency regulators, including OCT4, NANOG, and LIN28A. MATR3 is recruited with specific initiation factors on ribosomes, and controls pluripotency regulating the translation of specific transcripts including NANOG and LIN28A by direct binding and favouring their stabilization. Moreover, MATR3 stimulates the expression of YTHDF1 , which, in turn, binds the m6A-modified OCT4 mRNA. These results show that MATR3 orchestrates the pluripotency circuitry by modulating the translational efficiency and regulating the epitranscriptome of specific transcripts.

RNA-binding proteins (RBPs) are pleiotropic factors that control the processing and functional compartmentalization of transcripts by binding primarily to mRNA untranslated regions (UTRs). The competitive and/or cooperative interplay between RBPs and an array of coding and noncoding RNAs (ncRNAs) determines the posttranscriptional control of gene expression, influencing protein production. Recently, a variety of well-recognized and noncanonical RBP domains have been revealed by modern system-wide analyses, underlying an evolving classification of ribonucleoproteins (RNPs) and their importance in governing physiological RNA metabolism. The possibility of targeting selected RNA-protein interactions with small molecules is now expanding the concept of protein "druggability," with new implications for medicinal chemistry and for a deeper characterization of the mechanism of action of bioactive compounds. Here, taking SF3B1, HuR, LIN28, and Musashi proteins as paradigmatic case studies, we review the strategies applied for targeting RBPs, with emphasis on the technological advancements to study protein-RNA interactions and on the requirements of appropriate validation strategies to parallel high-throughput screening (HTS) efforts.

The human antigen R (HuR) is an RNA-binding protein known to modulate the expression of target mRNA coding for proteins involved in inflammation, tumorigenesis, and stress responses and is a valuable drug target. We previously found that dihydrotanshinone-I (DHTS, 1) prevents the association of HuR with its RNA substrate, thus imparing its function. Herein, inspired by DHTS structure, we designed and synthesized an array of ortho-quinones (tanshinone mimics) using a function-oriented synthetic approach. Among others, compound 6a and 6n turned out to be more effective than 1, showing a nanomolar Ki and disrupting HuR binding to RNA in cells. A combined approach of NMR titration and molecular dynamics (MD) simulations suggests that 6a stabilizes HuR in a peculiar closed conformation, which is incompatible with RNA binding. Alpha screen and RNA-electrophoretic mobility shift assays (REMSA) data on newly synthesized compounds allowed, for the first time, the generation of structure activity relationships (SARs), thus providing a solid background for the generation of highly effective HuR disruptors.

Cancer therapies are associated with increased infertility risk due to accelerated reproductive aging. Oxidative stress (OS) is a potential mechanism behind ovarian toxicity by cyclophosphamide (CPM), the most ovotoxic anticancer drug. An important sensor of OS is SIRT1, a NAD+-dependent deacetylase which regulates cellular defence and cell fate. This study investigated whether the natural carotenoid crocetin and the synthetic compound AS101 protect the ovary against CPM by modulating SIRT1 and mitochondrial markers. We found that the number of primordial follicles of female CD1 mice receiving crocetin plus CPM increased when compared with CPM alone and similar to AS101, whose protective effects are known. SIRT1 increased in CPM mouse ovaries revealing the occurrence of OS. Similarly, mitochondrial SIRT3 rose, whilst SOD2 and the mitochondrial biogenesis activator PGC1-α decreased, suggesting the occurrence of mitochondrial damage. Crocetin and AS101 administration prevented SIRT1 burst suggesting that preservation of redox balance can help the ovary to counteract ovarian damage by CPM. Decreased SIRT3 and increased SOD2 and PGC1-α in mice receiving crocetin or AS101 prior to CPM provide evidence for mitochondrial protection. Present results improve the knowledge of ovarian damage by CPM and may help to develop interventions for preserving fertility in cancer patients.

The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3′UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.