RESUMO
Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14's exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.
Assuntos
Metiltransferases , RNA Viral , SARS-CoV-2 , Proteínas não Estruturais Virais , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/química , Metiltransferases/metabolismo , Metiltransferases/genética , Metiltransferases/química , Metilação , RNA Viral/metabolismo , RNA Viral/química , RNA Viral/genética , Exorribonucleases/metabolismo , Exorribonucleases/genética , Humanos , Ligação Proteica , Capuzes de RNA/metabolismo , Capuzes de RNA/genética , Regulação Alostérica , COVID-19/virologia , COVID-19/genética , Multimerização Proteica , Replicação Viral/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/química , Proteínas Virais Reguladoras e AcessóriasRESUMO
It has become increasingly evident that the structures RNAs adopt are conformationally dynamic; the various structured states that RNAs sample govern their interactions with other nucleic acids, proteins, and ligands to regulate a myriad of biological processes. Although several biophysical approaches have been developed and used to study the dynamic landscape of structured RNAs, technical limitations have limited their application to all classes of RNA due to variable size and flexibility. Recent advances combining chemical probing experiments with next-generation- and direct sequencing have emerged as an alternative approach to exploring the conformational dynamics of RNA. In this review, we provide a methodological overview of the sequencing-based techniques used to study RNA conformational dynamics. We discuss how different techniques have enabled us to better understand the propensity of RNAs from a variety of different classes to sample multiple conformational states. Finally, we present examples of the ways these techniques have reshaped how we think about RNA structure.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Conformação de Ácido Nucleico , RNA , RNA/química , RNA/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nanoporos , Humanos , Análise de Sequência de RNA/métodosRESUMO
RNA's ability to form and interconvert between multiple secondary and tertiary structures is critical to its functional versatility and the traditional view of RNA structures as static entities has shifted towards understanding them as dynamic conformational ensembles. In this review we discuss RNA structural ensembles and their dynamics, highlighting the concept of conformational energy landscapes as a unifying framework for understanding RNA processes such as folding, misfolding, conformational changes, and complex formation. Ongoing advancements in cryo-electron microscopy and chemical probing techniques are significantly enhancing our ability to investigate multiple structures adopted by conformationally dynamic RNAs, while traditional methods such as nuclear magnetic resonance spectroscopy continue to play a crucial role in providing high-resolution, quantitative spatial and temporal information. We discuss how these methods, when used synergistically, can provide a comprehensive understanding of RNA conformational ensembles, offering new insights into their regulatory functions.
RESUMO
The centromere is a fundamental higher-order structure in chromosomes ensuring their faithful segregation upon cell division. Centromeric transcripts have been described in several species and suggested to participate in centromere function. However, low sequence conservation of centromeric repeats appears inconsistent with a role in recruiting highly conserved centromeric proteins. Here, we hypothesized that centromeric transcripts may function through a secondary structure rather than sequence conservation. Using mouse embryonic stem cells (ESCs), we show that an imbalance in the levels of forward or reverse minor satellite (MinSat) transcripts leads to severe chromosome segregation defects. We further show that MinSat RNA adopts a stem-loop secondary structure, which is conserved in human α-satellite transcripts. We identify an RNA binding region in CENPC and demonstrate that MinSat transcripts function through the structured region of the RNA. Importantly, mutants that disrupt MinSat secondary structure do not cause segregation defects. We propose that the conserved role of centromeric transcripts relies on their secondary RNA structure.