RESUMEN

Exportin 5 (Exp5, XPO5) is a nuclear export factor that functions in the microRNA (miRNA) biogenesis pathway to transport precursor miRNAs (pre-miRNAs) from the nucleus to the cytoplasm. Most of our current understanding of the Exp5 and pre-miRNA interaction is based on the investigation of how Exp5 binds to human pre-miR-30a (pre-miR-30 for short). As there are hundreds of human miRNA genes, how representative pre-miR-30 is, whether or how Exp5 interacts with distinct cargoes differentially, or whether Exp5 regulates miRNA expression, is unknown. Here we examined and compared the interactions between Exp5 and 157 human pre-miRNAs. We found that Exp5 binds distinct pre-miRNAs with modest variations in efficiencies, with the 3' overhang and the apical loop in pre-miRNAs being the two major discriminating factors. Exp5 binding efficiencies do not significantly correlate with endogenous miRNA expression, suggesting that Exp5 activity does not contribute to differential miRNA expression in vivo. Nonetheless, in human cells with reduced Exp5 levels, preferential Exp5 binding correlated with miRNA expression changes. Thus, our study provides a global picture of how Exp5 interacts with human pre-miRNAs and its role in regulating miRNA expression.Abbreviations: Exp5: Exportin 5; miRNA: microRNA; pri-miRNA: primary microRNA; pre-miRNA: precursor microRNA; nt: nucleotide.

Asunto(s)

Carioferinas/metabolismo , MicroARNs/genética , Precursores del ARN/genética , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Regulación de la Expresión Génica , Humanos , MicroARNs/química , MicroARNs/metabolismo , Conformación de Ácido Nucleico , Unión Proteica , Precursores del ARN/química , Precursores del ARN/metabolismo , Transporte de ARN , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes

RESUMEN

A large number of proteins involved in RNA metabolism possess a double-stranded RNA-binding domain (dsRBD), whose sequence variations and functional versatilities are still being recognized. All dsRBDs have a similar structural fold: α1-L1-ß1-L2-ß2-L3-ß3-L4-α2 (α represents an α-helix, ß a ß-sheet, and L a loop conformation between the well-defined secondary structures). Our recent work revealed that the dsRBD in Drosha, which is involved in animal microRNA (miRNA) biogenesis, differs from other dsRBDs by containing a short insertion in its L1 region and that this insertion is important for Drosha function. We asked why the same insertion is excluded in all other dsRBDs and proposed that a longer L1 may be detrimental to their functions. In this study, to test this hypothesis, we inserted the Drosha sequence into several well-known dsRBDs from various organisms. Gel mobility shift assay demonstrated that L1 extension invariably reduced RNA binding by these dsRBDs. In addition, such a mutation in Dicer, another protein involved in miRNA biogenesis, impaired Dicer's ability to process miRNAs, which led to de-repression of reporter expression, in human cells. Taken together, our results add to the growing appreciation of the diversity in dsRBDs and suggest that dsRBDs have intricate structures and functions that are sensitive to perturbations in the L1 region.

Asunto(s)

Motivo de Unión al ARN Bicatenario , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/fisiología , Secuencia de Aminoácidos , Animales , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/fisiología , ADN de Cadena Simple/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Células HEK293 , Humanos , MicroARNs/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/fisiología , Estructura Secundaria de Proteína , ARN/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleasa III/química , Ribonucleasa III/genética , Ribonucleasa III/fisiología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/fisiología

RESUMEN

In complex biochemical systems, an enzyme, protein, or RNA, symbolized as E, has hundreds or thousands of substrates or interacting partners. The relative specificity hypothesis proposes that such an E would differentially interact with and influence its many distinct, downstream substrates, thereby regulating the underlying biological process (es). The importance of relative specificity has been underappreciated, and evidence of its physiological consequences particularly lacking. Previously we showed that human Drosha and Dicer ribonucleases (RNases) both discriminate their respective microRNA (miRNA) substrates, and that differential cleavage by Drosha contributes to global differential miRNA expression. If relative specificity is an important biological mechanism, it should be evolutionarily conserved. To test this hypothesis, we hereby examined the cleavage of hundreds of zebrafish and fruitfly miRNA intermediates by Drosha and Dicer and the impact on miRNA biogenesis in these organisms. We showed that Drosha action regulates differential miRNA expression in zebrafish and fruitflies and identified the conserved secondary structure features and sequences in miRNA transcripts that control Drosha activity and miRNA expression. Our results established the conservation of miRNA processing mechanisms and regulatory functions by Drosha and Dicer, greatly strengthened the evidence for the physiological consequences of relative specificity as well as demonstrated its evolutionary significance.