RESUMO
The development of novel therapeutics that exploit alterations in the activation state of key cellular signaling pathways due to mutations in upstream regulators has generated the field of personalized medicine. These first-generation efforts have focused on actionable mutations identified by deep sequencing of large numbers of tumor samples. We propose that a second-generation opportunity exists by exploiting key downstream "nodes of control" that contribute to oncogenesis and are inappropriately activated due to loss of upstream regulation and microenvironmental influences. The RNA-binding protein HuR represents such a node. Because HuR functionality in cancer cells is dependent on HuR dimerization and its nuclear/cytoplasmic shuttling, we developed a new class of molecules targeting HuR protein dimerization. A structure-activity relationship algorithm enabled development of inhibitors of HuR multimer formation that were soluble, had micromolar activity, and penetrated the blood-brain barrier. These inhibitors were evaluated for activity validation and specificity in a robust cell-based assay of HuR dimerization. SRI-42127, a molecule that met these criteria, inhibited HuR multimer formation across primary patient-derived glioblastoma xenolines (PDGx), leading to arrest of proliferation, induction of apoptosis, and inhibition of colony formation. SRI-42127 had favorable attributes with central nervous system penetration and inhibited tumor growth in mouse models. RNA and protein analysis of SRI-42127-treated PDGx xenolines across glioblastoma molecular subtypes confirmed attenuation of targets upregulated by HuR. These results highlight how focusing on key attributes of HuR that contribute to cancer progression, namely cytoplasmic localization and multimerization, has led to the development of a novel, highly effective inhibitor. SIGNIFICANCE: These findings utilize a cell-based mechanism of action assay with a structure-activity relationship compound development pathway to discover inhibitors that target HuR dimerization, a mechanism required for cancer promotion.
Assuntos
Carcinogênese/efeitos dos fármacos , Proteína Semelhante a ELAV 1/química , Multimerização Proteica/efeitos dos fármacos , Algoritmos , Animais , Apoptose/efeitos dos fármacos , Barreira Hematoencefálica , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína Semelhante a ELAV 1/metabolismo , Proteína Semelhante a ELAV 1/fisiologia , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Camundongos , Camundongos Nus , Medicina de Precisão , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Ensaio Tumoral de Célula-Tronco , Regulação para CimaRESUMO
eIF3a is a putative subunit of the eukaryotic translation initiation factor 3 complex. Accumulating evidence suggests that eIF3a may have a translational regulatory function by suppressing translation of a subset of mRNAs while accelerating that of other mRNAs. Albeit the suppression of mRNA translation may derive from eIF3a binding to the 5'-UTRs of target mRNAs, how eIF3a may accelerate mRNA translation remains unknown. In this study, we show that eIF3a up-regulates translation of Chk1 but not Chk2 mRNA by interacting with HuR, which binds directly to the 3'-UTR of Chk1 mRNA. The interaction between eIF3a and HuR occurs at the 10-amino-acid repeat domain of eIF3a and the RNA recognition motif domain of HuR. This interaction may effectively circularize Chk1 mRNA to form an end-to-end complex that has recently been suggested to accelerate mRNA translation. Together with previous findings, we conclude that eIF3a may regulate mRNA translation by directly binding to the 5'-UTR to suppress or by interacting with RNA-binding proteins at 3'-UTRs to accelerate mRNA translation.
Assuntos
Quinase 1 do Ponto de Checagem/biossíntese , Proteína Semelhante a ELAV 1 , Fator de Iniciação 3 em Eucariotos , Biossíntese de Proteínas/fisiologia , Linhagem Celular , Proteína Semelhante a ELAV 1/química , Proteína Semelhante a ELAV 1/metabolismo , Fator de Iniciação 3 em Eucariotos/química , Fator de Iniciação 3 em Eucariotos/metabolismo , Regulação da Expressão Gênica , Humanos , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNARESUMO
The ELAVL1 (or human antigen R - HuR) RNA binding protein stabilizes the mRNA, with an AU-rich element, of several genes such as growth factors (i.e. VEGF) and inflammatory cytokines (i.e. TNFα). We hereby carried out a virtual screening campaign in order to identify and test novel HuR-mRNA disruptors. Best-scored compounds were tested in an in-vitro model of diabetic retinopathy, namely human retinal endothelial cells (HRECs) challenged with high-glucose levels (25 mM). HuR, VEGF and TNFα protein contents were evaluated by western-blot analysis in total cell lysates. VEGF and TNFα released from HRECs were measured in cell medium by ELISA. We found that two derivatives bearing indole moiety, VP12/14 and VP12/110, modulated HuR expression and decreased VEGF and TNF-α release by HREC exposed to high glucose (HG) levels. VP12/14 and VP12/110 inhibited VEGF and TNF-α release in HRECs challenged with high glucose levels, similarly to dihydrotanshinone (DHTS), a small molecule known to interfere with HuR- TNFα mRNA binding. The present findings demonstrated that VP12/14 and VP12/110 are innovative molecules with anti-inflammatory and anti-angiogenic properties, suggesting their potential use as novel candidates for treatment of diabetic retinopathy.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Células Endoteliais/metabolismo , Glucose/toxicidade , Indóis/administração & dosagem , RNA Mensageiro/metabolismo , Retina/metabolismo , Sítios de Ligação/fisiologia , Proteína Semelhante a ELAV 1/química , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Glucose/administração & dosagem , Humanos , Indóis/química , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Mensageiro/química , Retina/efeitos dos fármacos , Retina/patologiaRESUMO
Large bodies of studies have shown that the CRISPR/Cas9-based library screening is a very powerful tool for the identification of gene functions. However, most of these studies have focused on protein-coding genes, and, furthermore, very few studies have used gene reporters for screening. In the present study, we generated DNA methyltransferase 3B (DNMT3B) reporter and screened a CRISPR/Cas9 synergistic activation mediator (SAM) library against a focused group of lncRNAs. With this screening approach, we identified Rhabdomyosarcoma 2-Associated Transcript (RMST) as a positive regulator for DNMT3B. This was confirmed by activation of the endogenous RMST by SAM or ectopic expression of RMST. Moreover, RMST knockout (KO) suppresses DNMT3, while rescue with RMST in the KO cells restores the DNMT3 level. Finally, RMST KO suppresses global DNA methylation, leading to the upregulation of methylation-regulated genes. Mechanistically, RMST promotes the interaction between the RNA-binding protein HuR and DNMT3B 3' UTR, increasing the DNMT3B stability. Together, these results not only provide the feasibility of a reporter system for CRISPR library screening but also demonstrate the previously uncharacterized factor RMST as an important player in the modulation of DNA methylation.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , RNA Longo não Codificante/metabolismo , Regulação para Cima/genética , Regiões 3' não Traduzidas , Sistemas CRISPR-Cas , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Proteína Semelhante a ELAV 1/química , Estabilidade Enzimática/genética , Técnicas de Inativação de Genes , Genes Reporter , Células HCT116 , Células HEK293 , Humanos , Células MCF-7 , RNA Guia de Cinetoplastídeos/genética , RNA Longo não Codificante/genética , Transfecção , DNA Metiltransferase 3BRESUMO
Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate-rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3' untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12.
Assuntos
Proteínas ELAV/química , Proteína Semelhante a ELAV 1/química , Motivo de Reconhecimento de RNA/genética , Proteínas de Ligação a RNA/química , Regiões 3' não Traduzidas , Elementos Ricos em Adenilato e Uridilato/genética , Cristalografia por Raios X , Dimerização , Proteína Semelhante a ELAV 1/genética , Humanos , Espectroscopia de Ressonância Magnética , Proteínas de Ligação a RNA/genética , Ribonucleosídeo Difosfato Redutase/química , Proteínas Supressoras de Tumor/químicaRESUMO
In vitro and in vivo delivery of RNAs of interest holds promise for gene therapy. Recently, exosomes are considered as a kind of rational vehicle for RNA delivery, especially miRNA and/or siRNA, while the loading efficiency is limited. In this study, we engineered the exosomes for RNA loading by constructing a fusion protein in which the exosomal membrane protein CD9 was fused with RNA binding protein, while the RNA of interest either natively harbors or is engineered to have the elements for the binding. By proof-of-principle experiments, we here fused CD9 with HuR, an RNA binding protein interacting with miR-155 with a relatively high affinity. In the exosome packaging cells, the fused CD9-HuR successfully enriched miR-155 into exosomes when miR-155 was excessively expressed. Moreover, miR-155 encapsulated in the exosomes in turn could be efficiently delivered into the recipient cells and recognized the endogenous targets. In addition, we also revealed that the CD9-HuR exosomes could enrich the functional miRNA inhibitor or CRISPR/dCas9 when the RNAs were engineered to have the AU rich elements. Taken together, we here have established a novel strategy for enhanced RNA cargo encapsulation into engineered exosomes, which in turn functions in the recipient cells.
Assuntos
Proteína Semelhante a ELAV 1/química , Exossomos/química , MicroRNAs/química , Tetraspanina 29/química , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Proteína Semelhante a ELAV 1/genética , Exossomos/genética , Técnicas de Transferência de Genes , Humanos , Camundongos , MicroRNAs/genética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Tetraspanina 29/genéticaRESUMO
HuR/ELAVL1 is an RNA-binding protein involved in differentiation and stress response that acts primarily by stabilizing messenger RNA (mRNA) targets. HuR comprises three RNA recognition motifs (RRMs) where the structure and RNA binding of RRM3 and of full-length HuR remain poorly understood. Here, we report crystal structures of RRM3 free and bound to cognate RNAs. Our structural, NMR and biochemical data show that RRM3 mediates canonical RNA interactions and reveal molecular details of a dimerization interface localized on the α-helical face of RRM3. NMR and SAXS analyses indicate that the three RRMs in full-length HuR are flexibly connected in the absence of RNA, while they adopt a more compact arrangement when bound to RNA. Based on these data and crystal structures of tandem RRM1,2-RNA and our RRM3-RNA complexes, we present a structural model of RNA recognition involving all three RRM domains of full-length HuR. Mutational analysis demonstrates that RRM3 dimerization and RNA binding is required for functional activity of full-length HuR in vitro and to regulate target mRNAs levels in human cells, thus providing a fine-tuning for HuR activity in vivo.
Assuntos
Proteína Semelhante a ELAV 1/química , RNA/química , Linhagem Celular Tumoral , Proteína Semelhante a ELAV 1/metabolismo , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Multimerização Proteica , RNA/metabolismoRESUMO
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.
Assuntos
Proteína Semelhante a ELAV 1/química , Fenantrenos/química , Fenantrenos/farmacologia , Regiões 3' não Traduzidas , Elementos Ricos em Adenilato e Uridilato , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Furanos , Humanos , Espectroscopia de Ressonância Magnética , Camundongos Mutantes Neurológicos , Simulação de Dinâmica Molecular , Fenantrenos/metabolismo , Mutação Puntual , Conformação Proteica , Domínios Proteicos , Quinonas , RNA Mensageiro/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RNA-binding proteins (RBPs) and noncoding RNAs orchestrate post-transcriptional processes through the recognition of specific sites on targeted transcripts. Thus, understanding the connection between binding to specific sites and active regulation of the whole transcript is essential. Many immunoprecipitation techniques have been developed that identify either whole transcripts or binding sites of RBPs on each transcript using cell lysates. However, none of these methods simultaneously measures the strength of each binding site and quantifies binding to whole transcripts. In this study, we compare current procedures and present digestion optimized (DO)-RIP-seq, a simple method that locates and quantifies RBP binding sites using a continuous metric. We have used the RBP HuR/ELAVL1 to demonstrate that DO-RIP-seq can quantify HuR binding sites with high coverage across the entire human transcriptome, thereby generating metrics of relative RNA binding strength. We demonstrate that this quantitative enrichment of binding sites is proportional to the relative in vitro binding strength for these sites. In addition, we used DO-RIP-seq to quantify and compare HuR's binding to whole transcripts, thus allowing for seamless integration of binding site data with whole-transcript measurements. Finally, we demonstrate that DO-RIP-seq is useful for identifying functional mRNA target sets and binding sites where combinatorial interactions between HuR and AGO-microRNAs regulate the fate of the transcripts. Our data indicate that DO-RIP-seq will be useful for quantifying RBP binding events that regulate dynamic biological processes.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Perfilação da Expressão Gênica/métodos , MicroRNAs/genética , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Sítios de Ligação , Bases de Dados Genéticas , Proteína Semelhante a ELAV 1/química , Regulação da Expressão Gênica , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligação ProteicaRESUMO
AU-rich element binding/degradation factor 1 (AUF1) plays a role in destabilizing mRNAs by forming complexes with AU-rich elements (ARE) in the 3'-untranslated regions. Multiple AUF1-ARE complexes regulate the translation of encoded products related to the cell cycle, apoptosis, and inflammation. AUF1 contains two tandem RNA recognition motifs (RRM) and a Gln- (Q-) rich domain in their C-terminal region. To observe how the two RRMs are involved in recognizing ARE, we obtained the AUF1-p37 protein covering the two RRMs. However, only N-terminal RRM (RRM1) was crystallized and its structure was determined at 1.7 Å resolution. It appears that the RRM1 and RRM2 separated before crystallization. To demonstrate which factors affect the separate RRM1-2, we performed limited proteolysis using trypsin. The results indicated that the intact proteins were cleaved by unknown proteases that were associated with them prior to crystallization. In comparison with each of the monomers, the conformations of the ß2-ß3 loops were highly variable. Furthermore, a comparison with the RRM1-2 structures of HuR and hnRNP A1 revealed that a dimer of RRM1 could be one of the possible conformations of RRM1-2. Our data may provide a guidance for further structural investigations of AUF1 tandem RRM repeat and its mode of ARE binding.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo D/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Regiões 3' não Traduzidas , Apoptose , Cristalografia por Raios X , Proteína Semelhante a ELAV 1/química , Escherichia coli/metabolismo , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas/química , Humanos , Inflamação , Domínios Proteicos , Estrutura Secundária de Proteína , Proteólise , Estabilidade de RNA , Ribonucleases/química , Sequências de Repetição em TandemRESUMO
BACKGROUND: Targeted genomic editing using the CRISPR/Cas9 methodology has opened exciting new avenues in probing gene function in virtually any model system, including cultured mammalian cells. Depending on the desired mutation, several experimental options exist in the isolation of clonal lines, such as selection with introduced markers, or screening by PCR amplification of genomic DNA. However, streamlined approaches to establishing deletion and tagging mutants with minimal genomic perturbation are of interest in applying this methodology. RESULTS: We developed a procedure for rapid screening of clonal cell lines for the deletion of a protein of interest following CRISPR/Cas9 targeting in the absence of selective pressure based on dot immunoblots. To assess the technique, we probed clonal isolates of 293-TREx cells that were targeted with three separate sgRNAs against the HuR gene. Validation of knockout candidates by western blot indicated that the normalized protein abundances indicated by the dot blot serve as accurate predictors of deletion. In total, 32 independent biallelic deletion lines out of 248 screened clones were isolated, and recovery of null mutants ranged from 6 to 36% for the individual sgRNAs. Genomic sequencing verified small deletions at the targeted locus. CONCLUSIONS: Clonal screening for CRISPR/Cas9-mediated editing events using dot immunoblot is a straightforward and efficient approach that facilitates rapid generation of genomic mutants to study gene function.
Assuntos
Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Marcação de Genes , Immunoblotting , Sequência de Bases , Linhagem Celular , Proteína Semelhante a ELAV 1/química , Proteína Semelhante a ELAV 1/genética , Marcação de Genes/métodos , Genótipo , Humanos , Dados de Sequência Molecular , Mutação , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genética , Alinhamento de SequênciaRESUMO
The human antigen R (HuR) stabilizes many mRNAs of proto-oncogene, transcription factors, cytokines and growth factors by recognizing AU-rich elements (AREs) presented in their 3' or 5' untranslated region (UTR). Multiple lines of experimental evidence suggest that this process plays a key role in cancer development. Thus, destabilizing HuR/RNA interaction by small molecules presents an opportunity for cancer treatment/prevention. Here we present an integrated approach to identify inhibitors of HuR/RNA interaction using a combination of fluorescence-based and NMR-based high throughput screening (HTS). The HTS assay with fluorescence polarization readout and Z'-score of 0.8 was used to perform a screen of the NCI diversity set V library in a 384 well plate format. An NMR-based assay with saturation transfer difference (STD) detection was used for hits validation. Protein NMR spectroscopy was used to demonstrate that some hit compounds disrupt formation of HuR oligomer, whereas others block RNA binding. Thus, our integrated high throughput approach provides a new avenue for identification of small molecules targeting HuR/RNA interaction.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Polarização de Fluorescência/métodos , Espectroscopia de Ressonância Magnética/métodos , RNA/metabolismo , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Proteína Semelhante a ELAV 1/química , Ensaios de Triagem em Larga Escala/métodos , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Proto-Oncogene Mas , RNA/química , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Both RNA-binding proteins (RBP) and miRNA play important roles in the regulation of mRNA expression, often acting together to regulate a target mRNA. In some cases the RBP and miRNA have been reported to act competitively, but in other instances they function cooperatively. Here, we investigated HuR function as an enhancer of let-7-mediated translational repression of c-Myc despite the separation of their binding sites. Using an in vitro system, we determined that a let-7 mimic, consisting of single-stranded (ss)DNA complementary to the let-7 binding site, enhanced the affinity of HuR for a 122-nt MYC RNA encompassing both binding sites. This finding supports the biophysical principle of cooperative binding by an RBP and miRNA purely through interactions at distal mRNA binding sites.