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/metabolismoRESUMEN
RNA-binding proteins (RBPs) commonly feature multiple RNA-binding domains (RBDs), which provide these proteins with a modular architecture. Accumulating evidence supports that RBP architectural modularity and adaptability define the specificity of their interactions with RNA. However, how multiple RBDs recognize their cognate single-stranded RNA (ssRNA) sequences in concert remains poorly understood. Here, we use Upstream of N-Ras (Unr) as a model system to address this question. Although reported to contain five ssRNA-binding cold-shock domains (CSDs), we demonstrate that Unr includes an additional four CSDs that do not bind RNA (pseudo-RBDs) but are involved in mediating RNA tertiary structure specificity by reducing the conformational heterogeneity of Unr. Disrupting the interactions between canonical and non-canonical CSDs impacts RNA binding, Unr-mediated translation regulation, and the Unr-dependent RNA interactome. Taken together, our studies reveal a new paradigm in protein-RNA recognition, where interactions between RBDs and pseudo-RBDs select RNA tertiary structures, influence RNP assembly, and define target specificity.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Conformación de Ácido Nucleico , ARN/química , ARN/metabolismo , Secuencia de Aminoácidos , Animales , Drosophila melanogaster , Biosíntesis de Proteínas , Dominios ProteicosRESUMEN
Topological distributions of individual cellular clocks have not been demonstrated in peripheral organs. The cochlea displays circadian patterns of core clock gene expression [1, 2]. PER2 protein is expressed in the hair cells and spiral ganglion neurons of the cochlea in the spiral ganglion neurons [1]. To investigate the topological organization of cellular oscillators in the cochlea, we recorded circadian rhythms from mouse cochlear explants using highly sensitive real-time tracking of PER2::LUC bioluminescence. Here, we show cell-autonomous and self-sustained oscillations originating from hair cells and spiral ganglion neurons. Multi-phased cellular clocks were arranged along the length of the cochlea with oscillations initiating at the apex (low-frequency region) and traveling toward the base (high-frequency region). Phase differences of 3 hr were found between cellular oscillators in the apical and middle regions and from isolated individual cochlear regions, indicating that cellular networks organize the rhythms along the tonotopic axis. This is the first demonstration of a spatiotemporal arrangement of circadian clocks at the cellular level in a peripheral organ. Cochlear rhythms were disrupted in the presence of either voltage-gated potassium channel blocker (TEA) or extracellular calcium chelator (BAPTA), demonstrating that multiple types of ion channels contribute to the maintenance of coherent rhythms. In contrast, preventing action potentials with tetrodotoxin (TTX) or interfering with cell-to-cell communication the broad-spectrum gap junction blocker (CBX [carbenoxolone]) had no influence on cochlear rhythms. These findings highlight a dynamic regulation and longitudinal distribution of cellular clocks in the cochlea.