The Dual Organization of P-bodies Revealed by Immunoelectron Microscopy and Electron Tomography

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• 作者：Nicolas Cougot¹ ; ^{nicolas.cougot@univ-rennes1.fr} ; Annie Cavalier¹ ; Daniel Thomas¹ ; Reynald Gillet¹ ; ² ; ^{reynald.gillet@univ-rennes1.fr}
• 关键词：P-body ; processing body ; mRNP ; messenger ribonucleoprotein particle ; miRNA ; micro-RNA ; EDTA ; ethylenediaminetetraacetic acid ; rRNA ; ribosomal RNA ; TEM ; transmission electron microscopy ; 3D ; three-dimensional ; PBS ; phosphate-buffered saline ; DAPI ; 4&prime
• 刊名：Journal of Molecular Biology
• 出版年：2012
• 期刊代码：102_00222836
• 类别：imm
• 出版时间：29 June, 2012
• 卷：420
• 期：1-2
• 页码：17-28
• 文件大小：2998 K

摘要

Processing bodies (P-bodies) are cytoplasmic non-membranous domains involved in the regulation of eukaryotic gene expression. Since their discovery, several studies using fluorescence-based strategies have uncovered their pivotal role in mRNA metabolism, particularly during translation repression and/or mRNA degradation. Yet, P-bodies still remain a 鈥渂lack box鈥?in which numerous proteins accumulate next to RNAs to regulate their fate by unknown mechanisms. In this study, we investigated the ultrastructural organization of P-bodies in human cells. Using a wide range of original electron microscopy strategies, including high-pressure freezing and freeze substitution, we found that P-bodies are huge ribonucleoprotein complexes located in the close proximity of mitochondria and ribosomes, in which regulatory factors exhibit differential localization depending on their activity on mRNAs. We describe the first experiment pairing immunogold labeling with electron tomography (immunoelectron tomography) of a human P-body. Overall, the results depict a P-body organization that comprises at least two distinct compartments: a dense core on which peripheral protrusions are anchored.