RESUMEN
Many microRNAs regulate gene expression via atypical mechanisms, which are difficult to discern using native cross-linking methods. To ascertain the scope of non-canonical miRNA targeting, methods are needed that identify all targets of a given miRNA. We designed a new class of miR-CLIP probe, whereby psoralen is conjugated to the 3p arm of a pre-microRNA to capture targetomes of miR-124 and miR-132 in HEK293T cells. Processing of pre-miR-124 yields miR-124 and a 5'-extended isoform, iso-miR-124. Using miR-CLIP, we identified overlapping targetomes from both isoforms. From a set of 16 targets, 13 were differently inhibited at mRNA/protein levels by the isoforms. Moreover, delivery of pre-miR-124 into cells repressed these targets more strongly than individual treatments with miR-124 and iso-miR-124, suggesting that isomirs from one pre-miRNA may function synergistically. By mining the miR-CLIP targetome, we identified nine G-bulged target-sites that are regulated at the protein level by miR-124 but not isomiR-124. Using structural data, we propose a model involving AGO2 helix-7 that suggests why only miR-124 can engage these sites. In summary, access to the miR-124 targetome via miR-CLIP revealed for the first time how heterogeneous processing of miRNAs combined with non-canonical targeting mechanisms expand the regulatory range of a miRNA.
Asunto(s)
Proteínas Argonautas/metabolismo , Regulación de la Expresión Génica , MicroARNs/genética , Modelos Genéticos , Regiones no Traducidas 3'/genética , Secuencias de Aminoácidos , Proteínas Argonautas/química , Secuencia de Bases , Sitios de Unión , Biotina , Reactivos de Enlaces Cruzados/farmacología , ADN Complementario/genética , Proteínas de Unión al GTP/genética , Células HEK293 , Humanos , Inmunoprecipitación , MicroARNs/antagonistas & inhibidores , Proteínas Nucleares/genética , Conformación de Ácido Nucleico , Fotoquímica , Análisis de Secuencia de ADN , Estreptavidina , Trioxsaleno/efectos de la radiaciónRESUMEN
Huntington disease (HD) is a fatal neurodegenerative disorder caused by an expanded CAG repeat. Its length can be used to estimate the time of clinical diagnosis, which is defined by overt motor symptoms. Non-motor symptoms begin before motor onset, and involve changes in hypothalamus-regulated functions such as sleep, emotion and metabolism. Therefore we hypothesized that hypothalamic changes occur already prior to the clinical diagnosis. We performed voxel-based morphometry and logistic regression analyses of cross-sectional MR images from 220 HD gene carriers and 75 controls in the Predict-HD study. We show that changes in the hypothalamic region are detectable before clinical diagnosis and that its grey matter contents alone are sufficient to distinguish HD gene carriers from control cases. In conclusion, our study shows, for the first time, that alterations in grey matter contents in the hypothalamic region occur at least a decade before clinical diagnosis in HD using MRI.