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.

YTHDF proteins bind the N 6 -methyladenosine (m6A)-modified mRNAs, influencing their processing, stability, and translation. Therefore, the members of this protein family play crucial roles in gene regulation and several physiological and pathophysiological conditions. YTHDF proteins contain a hydrophobic pocket that accommodates the m6A embedded in the RRACH consensus sequence on mRNAs. We exploited the presence of this cage to set up an m6A-competitive assay and performed a high-throughput screen aimed at identifying ligands binding in the m6A pocket. We report the organoselenium compound ebselen as the first-in-class inhibitor of the YTHDF m6A-binding domain. Ebselen, whose interaction with YTHDF proteins was validated via orthogonal assays, cannot discriminate between the binding domains of the three YTHDF paralogs but can disrupt the interaction of the YTHDF m6A domain with the m6A-decorated mRNA targets. X-ray, mass spectrometry, and NMR studies indicate that in YTHDF1 ebselen binds close to the m6A cage, covalently to the Cys412 cysteine, or interacts reversibly depending on the reducing environment. We also showed that ebselen engages YTHDF proteins within cells, interfering with their mRNA binding. Finally, we produced a series of ebselen structural analogs that can interact with the YTHDF m6A domain, proving that ebselen expansion is amenable for developing new inhibitors. Our work demonstrates the feasibility of drugging the YTH domain in YTHDF proteins and opens new avenues for the development of disruptors of m6A recognition.

Summary 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 key pluripotency regulators' expression levels, including OCT4, NANOG, and LIN28A by pleiotropic mechanisms. MATR3 binds to the OCT4 and YTHDF1 promoters favoring their expression. YTHDF1, in turn, binds the m6A-modified OCT4 mRNA. Furthermore, MATR3 is recruited on ribosomes and controls pluripotency regulating the translation of specific transcripts, including NANOG and LIN28A, by direct binding and favoring their stabilization. These results show that MATR3 orchestrates the pluripotency circuitry by regulating the transcription, translational efficiency, and epitranscriptome of specific transcripts.

No abstract

Tauopathies are neurodegenerative disorders characterized by Tau aggregation. Genetic studies on familial cases allowed for the discovery of mutations in the MAPT gene that increase Tau propensity to detach from microtubules and to form insoluble cytoplasmic Tau aggregates. Recently, the rare mutation Q336H has been identified to be associated with Pick’s disease (PiD) and biochemical analyses demonstrated its ability to increase the microtubules (MTs) polymerization, thus revealing an opposite character compared to other Tau mutations studied so far. Here we investigated the biophysical and molecular properties of TauQ336H in living cells by the employment of the conformational Tau biosensor CST. We found that this mutation alters Tau conformation on microtubules, stabilizes its binding to tubulin, and is associated with a paradoxical lower level of Tau phosphorylation. Moreover, we found that this mutation impacts the cytoskeletal complexity by increasing the tubulin filament length and the number of branches. However, despite these apparently non-pathological traits, we observed the formation of intracellular inclusions confirming that Q336H leads to aggregation. Our results suggest that the Tau aggregation process might be triggered by molecular mechanisms other than Tau destabilization or post-translational modifications which are likely to be detrimental to neuronal function in vivo.

ELAV-like (ELAVL) RNA-binding proteins play a pivotal role in post-transcriptional processes, and their dysregulation is involved in several pathologies. This work was focused on HuD (ELAVL4), which is specifically expressed in nervous tissues, and involved in differentiation and synaptic plasticity mechanisms. HuD represents a new, albeit unexplored, candidate target for the treatment of several relevant neurodegenerative diseases. The aim of this pioneering work was the identification of new molecules able to recognize and bind HuD, thus interfering with its activity. We combined virtual screening, molecular dynamics (MD), and STD-NMR techniques. Starting from around 51 000 compounds, four promising hits eventually provided experimental evidence of their ability to bind HuD. Among the selected best hits, folic acid was found to be the most interesting one, being able to well recognize the HuD binding site. Our results provide a basis for the identification of new HuD interfering compounds which may be useful against neurodegenerative syndromes.

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 TMs in vitro but resulted to prodrug-like activity in 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 as Cxcl10, Il1b, Cd40, and Fas, 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, and in vivo in an LPS induced peritonitis model, in which the serum level of Cxcl10 and Il1b was strongly reduced, endowing TMs such as TM7nox with remarkable anti-inflammatory properties in vivo.

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 TMs in vitro but resulted to prodrug-like activity in 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 as Cxcl10, Il1b, Cd40, and Fas, 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, and in vivo in an LPS induced peritonitis model, in which the serum level of Cxcl10 and Il1b was strongly reduced, endowing TMs such as TM7nox with remarkable anti-inflammatory properties in vivo.