Continuous density gradients to study Argonaute and GW182 complexes associated with the endocytic pathway

Most complexes involved in RNA silencing were thought to be concentrated in cytoplasmic sites called P-bodies in the absence of stress. Accumulating evidence suggests that distinct cellular organelles or sites may be involved in the maturation of RNA-induced silencing complexes (RISC), decapping and deadenylation of miRNA-repressed mRNA, transport of translationally repressed mRNA, and disassembly of RISC complexes. Significant fractions of proteins essential for RNA silencing associate with membranes in general (GW182, AGO, and DICER), or more specifically with endoplasmic reticulum and Golgi (AGO), or endosomes and multivesicular bodies (AGO, GW182). In contrast, mRNA decapping and decay occur mainly in the cytoplasm. Continuous density gradients capable of partitioning these cellular compartments are valuable tools in efforts to decipher the complexes, trafficking and regulation of RISC throughout its biogenesis, action and turnover.