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See detailDemonstrating polymorphic miRNA-mediated gene regulation in vivo: application to the g+6223G->A mutation of Texel sheep.
Takeda, Haruko ULg; Charlier, Carole ULg; Farnir, Frédéric ULg et al

in RNA (New York, N.Y.) (2010), 16(9), 1854-63

We herein describe the development of a biochemical method to evaluate the effect of single nucleotide polymorphisms (SNPs) in target genes on their regulation by microRNAs in vivo. The method is based on ... [more ▼]

We herein describe the development of a biochemical method to evaluate the effect of single nucleotide polymorphisms (SNPs) in target genes on their regulation by microRNAs in vivo. The method is based on the detection of allelic imbalance in RNAs coimmunoprecipitated with AGO proteins from tissues of heterozygous individuals. We characterize the performances of our approach using a model system in a cell culture, and then apply it successfully to prove that the 3'UTR g+6223G-->A mutation operates by promoting RISC-dependent down-regulation of myostatin (MSTN) in skeletal muscle of Texel sheep. [less ▲]

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See detailDynamics and three-dimensional localization of ribosomal RNA within the nucleolus.
Thiry, Marc ULg; Cheutin, T.; O'Donohue, M. F. et al

in RNA (New York, N.Y.) (2000), 6(12), 1750-61

Although rRNA synthesis, maturation, and assembly into preribosomal particles occur within the nucleolus, the route taken by pre-rRNAs from their synthetic sites toward the cytoplasm remains largely ... [more ▼]

Although rRNA synthesis, maturation, and assembly into preribosomal particles occur within the nucleolus, the route taken by pre-rRNAs from their synthetic sites toward the cytoplasm remains largely unexplored. Here, we employed a nondestructive method for the incorporation of BrUTP into the RNA of living cells. By using pulse-chase experiments, three-dimensional image reconstructions of confocal optical sections, and electron microscopy analysis of ultrathin sections, we were able to describe topological and spatial dynamics of rRNAs within the nucleolus. We identified the precise location and the volumic organization of four typical subdomains, in which rRNAs are successively moving towards the nucleolar periphery during their synthesis and processing steps. The incorporation of BrUTP takes place simultaneously within several tiny spheres, centered on the fibrillar centers. Then, the structures containing the newly synthesized RNAs enlarge and appear as compact ringlets disposed around the fibrillar centers. Later, they form hollow spheres surrounding the latter components and begin to fuse together. Finally, these structures widen and form large rings reaching the limits of the nucleoli. These results clearly show that the transport of pre-rRNAs within the nucleolus does not occur randomly, but appears as a radial flow starting from the fibrillar centers that form concentric rings, which finally fuse together as they progress toward the nucleolar periphery. [less ▲]

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