RESUMO
Zinc finger (ZnF) domains appear in a pool of structural contexts and despite their small size achieve varying target specificities, covering single-stranded and double-stranded DNA and RNA as well as proteins. Combined with other RNA-binding domains, ZnFs enhance affinity and specificity of RNA-binding proteins (RBPs). The ZnF-containing immunoregulatory RBP Roquin initiates mRNA decay, thereby controlling the adaptive immune system. Its unique ROQ domain shape-specifically recognizes stem-looped cis-elements in mRNA 3'-untranslated regions (UTR). The N-terminus of Roquin contains a RING domain for protein-protein interactions and a ZnF, which was suggested to play an essential role in RNA decay by Roquin. The ZnF domain boundaries, its RNA motif preference and its interplay with the ROQ domain have remained elusive, also driven by the lack of high-resolution data of the challenging protein. We provide the solution structure of the Roquin-1 ZnF and use an RBNS-NMR pipeline to show that the ZnF recognizes AU-rich RNAs. We systematically refine the contributions of adenines in a poly(U)-background to specific complex formation. With the simultaneous binding of ROQ and ZnF to a natural target transcript of Roquin, our study for the first time suggests how Roquin integrates RNA shape and sequence features through the ROQ-ZnF tandem.
RESUMO
AT-rich interacting domain (ARID)-containing proteins, Arids, are a heterogeneous DNA-binding protein family involved in transcription regulation and chromatin processing. For the member Arid5a, no exact DNA-binding preference has been experimentally defined so far. Additionally, the protein binds to mRNA motifs for transcript stabilization, supposedly through the DNA-binding ARID domain. To date, however, no unbiased RNA motif definition and clear dissection of nucleic acid-binding through the ARID domain have been undertaken. Using NMR-centered biochemistry, we here define the Arid5a DNA preference. Further, high-throughput in vitro binding reveals a consensus RNA-binding motif engaged by the core ARID domain. Finally, transcriptome-wide binding (iCLIP2) reveals that Arid5a has a weak preference for (A)U-rich regions in pre-mRNA transcripts of factors related to RNA processing. We find that the intrinsically disordered regions flanking the ARID domain modulate the specificity and affinity of DNA binding, while they appear crucial for RNA interactions. Ultimately, our data suggest that Arid5a uses its extended ARID domain for bifunctional gene regulation and that the involvement of IDR extensions is a more general feature of Arids in interacting with different nucleic acids at the chromatin-mRNA interface.
Assuntos
Proteínas de Ligação a DNA , DNA , Fatores de Transcrição , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/química , DNA/metabolismo , DNA/química , DNA/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/química , Domínios Proteicos , Regulação da Expressão Gênica , Ligação Proteica , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA/metabolismo , RNA/química , RNA/genéticaRESUMO
The single-stranded RNA genome of SARS-CoV-2 is highly structured. Numerous helical stem-loop structures interrupted by mismatch motifs are present in the functionally important 5'- and 3'-UTRs. These mismatches modulate local helical geometries and feature unusual arrays of hydrogen bonding donor and acceptor groups. However, their conformational and dynamical properties cannot be directly inferred from chemical probing and are difficult to predict theoretically. A mismatch motif (SL1-motif) consisting of three consecutive Uâ¢U base pairs is located in stem-loop 1 of the 3'-UTR. We combined NMR-spectroscopy and MD-simulations to investigate its structure and dynamics. All three Uâ¢U base pairs feature two direct hydrogen bonds and are as stable as Watson-Crick A:U base pairs. Plasmodium falciparum 25S rRNA contains a triple Uâ¢U mismatch motif (Pf-motif) differing from SL1-motif only with respect to the orientation of the two closing base pairs. Interestingly, while the geometry of the outer two Uâ¢U mismatches was identical in both motifs the preferred orientation of the central Uâ¢U mismatch was different. MD simulations and potassium ion titrations revealed that the potassium ion-binding mode to the major groove is connected to the different preferred geometries of the central base pair in the two motifs.
Assuntos
Regiões 3' não Traduzidas , Pareamento Incorreto de Bases , Motivos de Nucleotídeos , RNA Viral , SARS-CoV-2 , Humanos , Pareamento de Bases , COVID-19/virologia , Genoma Viral , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Plasmodium falciparum/genética , RNA Viral/química , RNA Viral/genética , SARS-CoV-2/genética , SARS-CoV-2/químicaRESUMO
HnRNPs are ubiquitously expressed RNA-binding proteins, tightly controlling posttranscriptional gene regulation. Consequently, hnRNP networks are essential for cellular homeostasis and their dysregulation is associated with cancer and other diseases. However, the physiological function of hnRNPs in non-cancerous cell systems are poorly understood. We analyzed the importance of HNRNPDL in endothelial cell functions. Knockdown of HNRNPDL led to impaired proliferation, migration and sprouting of spheroids. Transcriptome analysis identified cyclin D1 (CCND1) and tropomyosin 4 (TPM4) as targets of HNRNPDL, reflecting the phenotypic changes after knockdown. Our findings underline the importance of HNRNPDL for the homeostasis of physiological processes in endothelial cells.
Assuntos
Células Endoteliais , Ribonucleoproteínas Nucleares Heterogêneas , Ribonucleoproteínas Nucleares Heterogêneas/genética , Células Endoteliais/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
We present the high-resolution structure of stem-loop 4 of the 5'-untranslated region (5_SL4) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) genome solved by solution state nuclear magnetic resonance spectroscopy. 5_SL4 adopts an extended rod-like structure with a single flexible looped-out nucleotide and two mixed tandem mismatches, each composed of a Gâ¢U wobble base pair and a pyrimidineâ¢pyrimidine mismatch, which are incorporated into the stem-loop structure. Both the tandem mismatches and the looped-out residue destabilize the stem-loop structure locally. Their distribution along the 5_SL4 stem-loop suggests a role of these non-canonical elements in retaining functionally important structural plasticity in particular with regard to the accessibility of the start codon of an upstream open reading frame located in the RNA's apical loop. The apical loop-although mostly flexible-harbors residual structural features suggesting an additional role in molecular recognition processes. 5_SL4 is highly conserved among the different variants of SARS-CoV-2 and can be targeted by small molecule ligands, which it binds with intermediate affinity in the vicinity of the non-canonical elements within the stem-loop structure.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Sequência de Bases , COVID-19/virologia , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , RNA Viral/química , SARS-CoV-2/química , SARS-CoV-2/genéticaRESUMO
The outbreak of COVID-19 in December 2019 required the formation of international consortia for a coordinated scientific effort to understand and combat the virus. In this Viewpoint Article, we discuss how the NMR community has gathered to investigate the genome and proteome of SARS-CoV-2 and tested them for binding to low-molecular-weight binders. External factors including extended lockdowns due to the global pandemic character of the viral infection triggered the transition from locally focused collaborative research conducted within individual research groups to digital exchange formats for immediate discussion of unpublished results and data analysis, sample sharing, and coordinated research between more than 50 groups from 18 countries simultaneously. We discuss key lessons that might pertain after the end of the pandemic and challenges that we need to address.
Assuntos
COVID-19 , Humanos , SARS-CoV-2 , Controle de Doenças Transmissíveis , Espectroscopia de Ressonância Magnética , Imageamento por Ressonância MagnéticaRESUMO
The ongoing pandemic of the respiratory disease COVID-19 is caused by the SARS-CoV-2 (SCoV2) virus. SCoV2 is a member of the Betacoronavirus genus. The 30 kb positive sense, single stranded RNA genome of SCoV2 features 5'- and 3'-genomic ends that are highly conserved among Betacoronaviruses. These genomic ends contain structured cis-acting RNA elements, which are involved in the regulation of viral replication and translation. Structural information about these potential antiviral drug targets supports the development of novel classes of therapeutics against COVID-19. The highly conserved branched stem-loop 5 (SL5) found within the 5'-untranslated region (5'-UTR) consists of a basal stem and three stem-loops, namely SL5a, SL5b and SL5c. Both, SL5a and SL5b feature a 5'-UUUCGU-3' hexaloop that is also found among Alphacoronaviruses. Here, we report the extensive 1H, 13C and 15N resonance assignment of the 37 nucleotides (nts) long sequence spanning SL5b and SL5c (SL5b + c), as basis for further in-depth structural studies by solution NMR spectroscopy.
Assuntos
COVID-19 , SARS-CoV-2 , Regiões 5' não Traduzidas , Humanos , Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear BiomolecularRESUMO
The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7-33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.
Assuntos
Regiões 5' não Traduzidas , Ressonância Magnética Nuclear Biomolecular , SARS-CoV-2/genética , Conformação de Ácido Nucleico , RNA Líder para ProcessamentoRESUMO
Posttranscriptional regulation at mRNA level is defined not only by sequence, but also by the structure of the mRNA. Structures in the untranslated regions control stability, translation and localization of an mRNA. Evolutionary conservation can be used to identify such regulatory active structures in mRNAs. For example, constitutive decay element (CDE) stem-loops are recognized by Roquin proteins in an exclusively shape-specific manner. Strikingly, some CDEs can serve a dual function in gene repression depending on their folding status.
RESUMO
SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1 H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.
Assuntos
Genoma , RNA Viral/metabolismo , SARS-CoV-2/genética , Bibliotecas de Moléculas Pequenas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ligantes , Estrutura Molecular , Conformação de Ácido Nucleico , Espectroscopia de Prótons por Ressonância Magnética , RNA Viral/química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
The SARS-CoV-2 virus is the cause of the respiratory disease COVID-19. As of today, therapeutic interventions in severe COVID-19 cases are still not available as no effective therapeutics have been developed so far. Despite the ongoing development of a number of effective vaccines, therapeutics to fight the disease once it has been contracted will still be required. Promising targets for the development of antiviral agents against SARS-CoV-2 can be found in the viral RNA genome. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are highly conserved among Betacoronaviruses and contain structured RNA elements involved in the translation and replication of the viral genome. The 40 nucleotides (nt) long highly conserved stem-loop 4 (5_SL4) is located within the 5'-untranslated region (5'-UTR) important for viral replication. 5_SL4 features an extended stem structure disrupted by several pyrimidine mismatches and is capped by a pentaloop. Here, we report extensive 1H, 13C, 15N and 31P resonance assignments of 5_SL4 as the basis for in-depth structural and ligand screening studies by solution NMR spectroscopy.
Assuntos
Regiões 5' não Traduzidas , Ressonância Magnética Nuclear Biomolecular , SARS-CoV-2/genética , Sequências Repetidas Invertidas/genéticaRESUMO
Proteome integrity is crucial for cellular homeostasis and adaptation to stress conditions such as hypoxia. One mechanism for rapid adaptation of the proteome in response to changing environmental signals is alternative splicing. In addition to generating different protein isoforms, alternative splicing is also capable of controlling total protein levels by the regulated synthesis of non-productive mRNA isoforms. The hypoxia-induced isoform E of the tumor suppressor MAX is produced by retention and translation of the last intron. This leads to an alternative C-terminus that harbors a potent C-degron, the isoE degron. Strikingly, the isoE degron represents a universal protein degradation signal that is not only functional in mammalian cells, but also in yeast and even in bacteria. Essential for efficient protein decay is a conserved (F/W)xxW motif. Degradation of isoE tagged proteins is mediated by the proteasome in eukaryotes and Lon protease in bacteria. Thus, the isoE degron is a broadly applicable and highly efficient tool in protein analyses.
Assuntos
Processamento Alternativo , Proteólise , Proteoma , Bactérias , Escherichia coli , Eucariotos , Células HeLa , Humanos , Hipóxia , Íntrons , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/metabolismo , Saccharomyces cerevisiaeRESUMO
The SARS-CoV-2 (SCoV-2) virus is the causative agent of the ongoing COVID-19 pandemic. It contains a positive sense single-stranded RNA genome and belongs to the genus of Betacoronaviruses. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are potential antiviral drug targets. Major parts of these sequences are highly conserved among Betacoronaviruses and contain cis-acting RNA elements that affect RNA translation and replication. The 31 nucleotide (nt) long highly conserved stem-loop 5a (SL5a) is located within the 5'-untranslated region (5'-UTR) important for viral replication. SL5a features a U-rich asymmetric bulge and is capped with a 5'-UUUCGU-3' hexaloop, which is also found in stem-loop 5b (SL5b). We herein report the extensive 1H, 13C and 15N resonance assignment of SL5a as basis for in-depth structural studies by solution NMR spectroscopy.
Assuntos
Regiões 5' não Traduzidas , Proteases Semelhantes à Papaína de Coronavírus/química , Espectroscopia de Ressonância Magnética , SARS-CoV-2/química , SARS-CoV-2/genética , Isótopos de Carbono , Genes Virais , Hidrogênio , Isótopos de Nitrogênio , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de ProteínaRESUMO
The current outbreak of the highly infectious COVID-19 respiratory disease is caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). To fight the pandemic, the search for promising viral drug targets has become a cross-border common goal of the international biomedical research community. Within the international Covid19-NMR consortium, scientists support drug development against SARS-CoV-2 by providing publicly available NMR data on viral proteins and RNAs. The coronavirus nucleocapsid protein (N protein) is an RNA-binding protein involved in viral transcription and replication. Its primary function is the packaging of the viral RNA genome. The highly conserved architecture of the coronavirus N protein consists of an N-terminal RNA-binding domain (NTD), followed by an intrinsically disordered Serine/Arginine (SR)-rich linker and a C-terminal dimerization domain (CTD). Besides its involvement in oligomerization, the CTD of the N protein (N-CTD) is also able to bind to nucleic acids by itself, independent of the NTD. Here, we report the near-complete NMR backbone chemical shift assignments of the SARS-CoV-2 N-CTD to provide the basis for downstream applications, in particular site-resolved drug binding studies.
Assuntos
Proteínas do Nucleocapsídeo de Coronavírus/química , Espectroscopia de Ressonância Magnética , SARS-CoV-2/química , Isótopos de Carbono , Cristalografia por Raios X , Dimerização , Desenho de Fármacos , Hidrogênio , Concentração de Íons de Hidrogênio , Isótopos de Nitrogênio , Fosfoproteínas/química , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Estrutura Secundária de ProteínaRESUMO
The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.
Assuntos
COVID-19/prevenção & controle , Espectroscopia de Ressonância Magnética/métodos , Conformação de Ácido Nucleico , RNA Viral/química , SARS-CoV-2/genética , Regiões 3' não Traduzidas/genética , Sequência de Bases , COVID-19/epidemiologia , COVID-19/virologia , Mudança da Fase de Leitura do Gene Ribossômico/genética , Genoma Viral/genética , Humanos , Modelos Moleculares , Pandemias , SARS-CoV-2/fisiologiaRESUMO
The ongoing pandemic caused by the Betacoronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) demonstrates the urgent need of coordinated and rapid research towards inhibitors of the COVID-19 lung disease. The covid19-nmr consortium seeks to support drug development by providing publicly accessible NMR data on the viral RNA elements and proteins. The SARS-CoV-2 genome encodes for approximately 30 proteins, among them are the 16 so-called non-structural proteins (Nsps) of the replication/transcription complex. The 217-kDa large Nsp3 spans one polypeptide chain, but comprises multiple independent, yet functionally related domains including the viral papain-like protease. The Nsp3e sub-moiety contains a putative nucleic acid-binding domain (NAB) with so far unknown function and consensus target sequences, which are conceived to be both viral and host RNAs and DNAs, as well as protein-protein interactions. Its NMR-suitable size renders it an attractive object to study, both for understanding the SARS-CoV-2 architecture and drugability besides the classical virus' proteases. We here report the near-complete NMR backbone chemical shifts of the putative Nsp3e NAB that reveal the secondary structure and compactness of the domain, and provide a basis for NMR-based investigations towards understanding and interfering with RNA- and small-molecule-binding by Nsp3e.
Assuntos
Betacoronavirus/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Isótopos de Nitrogênio/química , Ácidos Nucleicos/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Proteínas não Estruturais Virais/química , Ligação Proteica , Domínios Proteicos , SARS-CoV-2RESUMO
Adenylate/uridylate-rich elements (AREs) are the most common cis-regulatory elements in the 3'-untranslated region (UTR) of mRNAs, where they fine-tune turnover by mediating mRNA decay. They increase plasticity and efficacy of mRNA regulation and are recognized by several ARE-specific RNA-binding proteins (RBPs). Typically, AREs are short linear motifs with a high content of complementary A and U nucleotides and often occur in multiple copies. Although thermodynamically rather unstable, the high AU-content might enable transient secondary structure formation and modify mRNA regulation by RBPs. We have recently suggested that the immunoregulatory RBP Roquin recognizes folded AREs as constitutive decay elements (CDEs), resulting in shape-specific ARE-mediated mRNA degradation. However, the structural evidence for a CDE-like recognition of AREs by Roquin is still lacking. We here present structures of CDE-like folded AREs, both in their free and protein-bound form. Moreover, the AREs in the UCP3 3'-UTR are additionally bound by the canonical ARE-binding protein AUF1 in their linear form, adopting an alternative binding-interface compared to the recognition of their CDE structure by Roquin. Strikingly, our findings thus suggest that AREs can be recognized in multiple ways, allowing control over mRNA regulation by adapting distinct conformational states, thus providing differential accessibility to regulatory RBPs.
Assuntos
Elementos Ricos em Adenilato e Uridilato , Proteínas de Ligação a RNA/química , Ubiquitina-Proteína Ligases/química , Sítios de Ligação , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Motivos de Nucleotídeos , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Hypoxia is a hallmark of solid cancers, supporting proliferation, angiogenesis, and escape from apoptosis. There is still limited understanding of how cancer cells adapt to hypoxic conditions and survive. We analyzed transcriptome changes of human lung and breast cancer cells under chronic hypoxia. Hypoxia induced highly concordant changes in transcript abundance, but divergent splicing responses, underlining the cell type-specificity of alternative splicing programs. While RNA-binding proteins were predominantly reduced, hypoxia specifically induced muscleblind-like protein 2 (MBNL2). Strikingly, MBNL2 induction was critical for hypoxia adaptation by controlling the transcript abundance of hypoxia response genes, such as vascular endothelial growth factor A (VEGFA) MBNL2 depletion reduced the proliferation and migration of cancer cells, demonstrating an important role of MBNL2 as cancer driver. Hypoxia control is specific for MBNL2 and not shared by its paralog MBNL1. Thus, our study revealed MBNL2 as central mediator of cancer cell responses to hypoxia, regulating the expression and alternative splicing of hypoxia-induced genes.
Assuntos
Neoplasias da Mama/genética , Neoplasias Pulmonares/genética , Proteínas de Ligação a RNA/genética , Hipóxia Tumoral/genética , Fator A de Crescimento do Endotélio Vascular/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Transcriptoma/genéticaRESUMO
Hypoxia is associated with several diseases, including cancer. Cells that are deprived of adequate oxygen supply trigger transcriptional and post-transcriptional responses, which control cellular pathways such as angiogenesis, proliferation, and metabolic adaptation. Circular RNAs (circRNAs) are a novel class of mainly non-coding RNAs, which have been implicated in multiple cancers and attract increasing attention as potential biomarkers. Here, we characterize the circRNA signatures of three different cancer cell lines from cervical (HeLa), breast (MCF-7), and lung (A549) cancer under hypoxia. In order to reliably detect circRNAs, we integrate available tools with custom approaches for quantification and statistical analysis. Using this consolidated computational pipeline, we identify ~12000 circRNAs in the three cancer cell lines. Their molecular characteristics point to an involvement of complementary RNA sequences as well as trans-acting factors in circRNA biogenesis, such as the RNA-binding protein HNRNPC. Notably, we detect a number of circRNAs that are more abundant than their linear counterparts. In addition, 64 circRNAs significantly change in abundance upon hypoxia, in most cases in a cell type-specific manner. In summary, we present a comparative circRNA profiling in human cancer cell lines, which promises novel insights into the biogenesis and function of circRNAs under hypoxic stress.