10.6084/m9.figshare.7928624.v1
Andrea Marranci
Andrea
Marranci
Romina D’Aurizio
Romina
D’Aurizio
Sebastian Vencken
Sebastian
Vencken
Serena Mero
Serena
Mero
Elena Guzzolino
Elena
Guzzolino
Milena Rizzo
Milena
Rizzo
Letizia Pitto
Letizia
Pitto
Marco Pellegrini
Marco
Pellegrini
Giovanna Chiorino
Giovanna
Chiorino
Catherine M. Greene
Catherine M.
Greene
Laura Poliseno
Laura
Poliseno
Systematic evaluation of the microRNAome through miR-CATCHv2.0 identifies positive and negative regulators of <i>BRAF</i>-X1 mRNA
Taylor & Francis Group
2019
Direct microRNA-target binding
affinity purification
BRAF-X1 mRNA
transcript stability
transcript translation
ERK signalling
melanoma
2019-03-30 16:03:00
Dataset
https://tandf.figshare.com/articles/dataset/Systematic_evaluation_of_the_microRNAome_through_miR-CATCHv2_0_identifies_positive_and_negative_regulators_of_i_BRAF_i_-X1_mRNA/7928624
<p>Here we present miR-CATCHv2.0, an implemented experimental method that allows the identification of the microRNA species directly bound to an RNA of interest. After cross-linking of microRNA::RNA::Ago2 complexes using formaldehyde, the RNA is fragmented using sonication and then subjected to affinity purification using two sets of biotinylated tiling probes (ODD and EVEN). Finally, enriched microRNA species are retrieved by means of small RNA sequencing coupled with an <i>ad hoc</i> analytical workflow.</p> <p>In BRAFV600E mutant A375 melanoma cells, miR-CATCHv2.0 allowed us to identify 20 microRNAs that target X1, the most abundant isoform of <i>BRAF</i> mRNA. These microRNAs fall into different functional classes, according to the effect that they exert (decrease/increase in BRAFV600E mRNA and protein levels) and to the mechanism they use to achieve it (destabilization/stabilization of X1 mRNA or decrease/increase in its translation). microRNA-induced variations in BRAFV600E protein levels are most of the times coupled to consistent variations in pMEK levels, in melanoma cell proliferation <i>in vitro</i> and in sensitivity to the BRAF inhibitor vemurafenib in a xenograft model in zebrafish. However, microRNAs exist that uncouple the degree of activation of the ERK pathway from the levels of BRAFV600E protein.</p> <p>Our study proposes miR-CATCHv2.0 as an effective tool for the identification of direct microRNA-target interactions and, by using such a tool, unveils the complexity of the post-transcriptional regulation to which BRAFV600E and the ERK pathway are subjected in melanoma cells.</p>