MicroRNA-101 expression is associated with JAK2V617F activity and regulates JAK2/STAT5 signaling.

Philadelphia negative myeloproliferative neopl 28 asms (MPNs) are clonal haematological diseases characterized by excessive production of mature blood cells. Exome sequencing of patient samples have showed a relatively low degree genomic complexity for these diseases1. The majority of MPN patients carry somatic mutations in the JAK2 gene, with the JAK2V617F missense mutation being the most common in poly33 cythemia vera (PV, 95%) and essential thrombocythemia (ET, 60%) 2.FP was supported by Fondazione Umberto Veronesi, and Institute Pasteur - Fondazione Cenci Bolognetti

Kinetic modelling of competition and depletion of shared miRNAs by competing endogenous RNAs

Non-conding RNAs play a key role in the post-transcriptional regulation of mRNA translation and turnover in eukaryotes. miRNAs, in particular, interact with their target RNAs through protein-mediated, sequence-specific binding, giving rise to extended and highly heterogeneous miRNA-RNA interaction networks. Within such networks, competition to bind miRNAs can generate an effective positive coupling between their targets. Competing endogenous RNAs (ceRNAs) can in turn regulate each other through miRNA-mediated crosstalk. Albeit potentially weak, ceRNA interactions can occur both dynamically, affecting e.g. the regulatory clock, and at stationarity, in which case ceRNA networks as a whole can be implicated in the composition of the cell's proteome. Many features of ceRNA interactions, including the conditions under which they become significant, can be unraveled by mathematical and in silico models. We review the understanding of the ceRNA effect obtained within such frameworks, focusing on the methods employed to quantify it, its role in the processing of gene expression noise, and how network topology can determine its reach.Comment: review article, 29 pages, 7 figure

Multiple mechanisms disrupt the let-7 microRNA family in neuroblastoma

Poor prognosis in neuroblastoma is associated with genetic amplification of MYCN. MYCN is itself a target of let-7, a tumour suppressor family of microRNAs implicated in numerous cancers. LIN28B, an inhibitor of let-7 biogenesis, is overexpressed in neuroblastoma and has been reported to regulate MYCN. Here we show, however, that LIN28B is dispensable in MYCN-amplified neuroblastoma cell lines, despite de-repression of let-7. We further demonstrate that MYCN messenger RNA levels in amplified disease are exceptionally high and sufficient to sponge let-7, which reconciles the dispensability of LIN28B. We found that genetic loss of let-7 is common in neuroblastoma, inversely associated with MYCN amplification, and independently associated with poor outcomes, providing a rationale for chromosomal loss patterns in neuroblastoma. We propose that let-7 disruption by LIN28B, MYCN sponging, or genetic loss is a unifying mechanism of neuroblastoma development with broad implications for cancer pathogenesis.United States. National Institutes of Health (R01GM107536)Alex's Lemonade Stand FoundationHoward Hughes Medical InstituteBoston Children's Hospital. Manton Center for Orphan Disease ResearchNational Institute of General Medical Sciences (U.S.) (T32GM007753

Enhanced Probe-Based RT-qPCR Quantification of MicroRNAs Using Poly(A) Tailing and 5′ Adaptor Ligation

International audienceProbe-based quantitative PCR (qPCR) is a commonly used tool in the realm of real-time qPCR experiments since it is one of the most sensitive detection methods allowing an accurate and reproducible analysis. It uses real-time fluorescence from a fluorescently labeled probe that specifically targets the desired PCR product to measure DNA amplification at each cycle of the PCR. Coupled to a proper reverse transcription step, probe-based qPCR can be efficiently used for the analysis of the expression of difficult targets such as miRNAs. In this chapter, we describe the TaqMan® advanced miRNA assay in which, owing to a poly(A)-tailing step, the reverse transcription is advantageously performed at once for all the miRNAs in a given sample, and, coupled to the ligation of a 5' universal adapter, allows for a supplementary pre-qPCR amplification step increasing the sensitivity of the assay. Along this protocol, we also provide our general guidelines and advices to perform a reliable and successful quantitative analysis

iCLIP of the PIWI Protein Aubergine in Drosophila Embryos.

International audiencePiwi-interacting RNAs (piRNAs) are a class of small noncoding RNAs bound to specific Argonaute proteins, the PIWI proteins. piRNAs target mRNAs by complementarity to silence them; they play an important role in the repression of transposable elements in the germ line of many species. piRNAs and PIWI proteins are also involved in diverse biological processes through their role in the regulation of cellular mRNAs. In the Drosophila embryo, they contribute to the maternal mRNA decay occurring during the maternal-to-zygotic transition. CLIP (UV cross-linking and immunoprecipitation) techniques have been used to identify target mRNAs of Argonaute proteins. Here we describe the iCLIP (individual-nucleotide resolution CLIP) protocol that we have adapted for the PIWI protein Aubergine in Drosophila embryos