The packaging of cytoplasmic mRNA into discrete RNA granules regulates gene expression by delaying the translation of specific transcripts. Specialized RNA granules found in germ cells direct the timing of maternal mRNA translation to promote germ cell development in the early embryo and establish the germ line for the next generation. Similarly, select neuronal mRNA transcripts are packaged into translationally inert RNA granules, transported to sites where their protein products are required, and only then activated and translated. Following translation, however, newly inactivated mRNAs released from polysomes can also be packaged into dynamic, transient structures known as stress granules (SGs) and processing bodies (PBs). Stress granules are composed largely of stalled preinitiation complexes, and contain mRNA, small ribosomal subunits, eIF3, eIF4E, eIF4G, and PABP, as their core components. PBs are associated with mRNA decay and contain the decapping enzymes DCP1/2, the 5′ to 3′ exonuclease Xrn1, the Lsm proteins (1-7), and the scaffolding proteins hedls/GE-1 and GW182. Both SGs and PBs contain mRNA, eIF4E, microRNAs and argonaute proteins, and various regulators of mRNA stability and translation (TTP, RCK/p54, and CPEB). Thus, SGs and PBs share some protein and mRNA components, but also contain a number of unique markers specific to each structure. We describe markers and staining procedures used to identify these distinct types of RNA granules, describe conditions that promote their assembly and disassembly, and establish YB-1 as a useful marker of SGs and PBs. © 2007 Elsevier Inc. All rights reserved.
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