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1.
RNA ; 29(3): 317-329, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36617673

RESUMEN

RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use RNA:RNA binding by SHAPE (RABS) to investigate microRNA-34a (miR-34a) binding its mRNA target, the silent information regulator 1 (mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.


Asunto(s)
MicroARNs , MicroARNs/genética , MicroARNs/metabolismo , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/metabolismo , Interferencia de ARN , Proteínas Argonautas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo
2.
Nucleic Acids Res ; 51(20): 11162-11177, 2023 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-37819016

RESUMEN

MicroRNAs (miRNAs) are short RNAs that post-transcriptionally regulate gene expression by binding to specific sites in mRNAs. Site recognition is primarily mediated by the seed region (nucleotides g2-g8 in the miRNA), but pairing beyond the seed (3'-pairing) is important for some miRNA:target interactions. Here, we use SHAPE, luciferase reporter assays and transcriptomics analyses to study the combined effect of 3'-pairing and secondary structures in mRNAs on repression efficiency. Using the interaction between miR-34a and its SIRT1 binding site as a model, we provide structural and functional evidence that 3'-pairing can compensate for low seed-binding site accessibility, enabling repression of sites that would otherwise be ineffective. We show that miRNA 3'-pairing regions can productively base-pair with nucleotides far upstream of the seed-binding site and that both hairpins and unstructured bulges within the target site are tolerated. We use SHAPE to show that sequences that overcome inaccessible seed-binding sites by strong 3'-pairing adopt the predicted structures and corroborate the model using luciferase assays and high-throughput modelling of 8177 3'-UTR targets for six miRNAs. Finally, we demonstrate that PHB2, a target of miR-141, is an inaccessible target rescued by efficient 3'-pairing. We propose that these results could refine predictions of effective target sites.


Asunto(s)
MicroARNs , ARN Mensajero , Emparejamiento Base , Luciferasas/genética , MicroARNs/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Regiones no Traducidas 3' , Regulación de la Expresión Génica , Conformación de Ácido Nucleico
3.
Nucleic Acids Res ; 48(22): 12415-12435, 2020 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-33167030

RESUMEN

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.


Asunto(s)
COVID-19/prevención & control , Espectroscopía de Resonancia Magnética/métodos , Conformación de Ácido Nucleico , ARN Viral/química , SARS-CoV-2/genética , Regiones no Traducidas 3'/genética , Secuencia de Bases , COVID-19/epidemiología , COVID-19/virología , Sistema de Lectura Ribosómico/genética , Genoma Viral/genética , Humanos , Modelos Moleculares , Pandemias , SARS-CoV-2/fisiología
5.
MethodsX ; 9: 101861, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36187158

RESUMEN

The ability to describe the topology of a folded protein conformation is critically important for functional analysis, protein engineering, and drug design. Circuit topology is a unique topological framework which is widely applicable to protein analysis, yet a state-of-the art implementation of this concept is lacking. Here, we present an open-source Python-implemented circuit topology tool called ProteinCT. The platform provides a method for acquiring, visualizing, analyzing, and quantifying circuit topology data from proteins of interest. We mapped the universe of human proteins to a circuit topology space using conventional hardware within a few hours, demonstrating the performance of ProteinCT. In brief,•A Python-implemented circuit topology tool is developed to extract global and local topological information from a protein structure file.•Modules are developed to combine topological information with geometric and energetic information.•It is demonstrated that the method can be efficiently applied to a large set of proteins, opening a wide range of possibilities for structural proteomics research.

6.
Biomol NMR Assign ; 15(1): 203-211, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33484403

RESUMEN

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.


Asunto(s)
Regiones no Traducidas 5' , Proteasas Similares a la Papaína de Coronavirus/química , Espectroscopía de Resonancia Magnética , SARS-CoV-2/química , SARS-CoV-2/genética , Isótopos de Carbono , Genes Virales , Hidrógeno , Isótopos de Nitrógeno , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína
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