Characterization of cytoplasmic bodies involved in 5' to 3' mRNA degradation in human cells

Abstract

In the context of gene expression regulation, discontinuing translation of mRNAs occurs by evolutionarily conserved mechanisms whose executors have been described to be associated with various types of cytoplasmic mRNA granules. Processing (P) bodies are such granules where mRNA co-localizes with effectors of the 5' to 3' mRNA degradation, RNA interference and Nonsense Mediated Decay pathways. In this manuscript we demonstrate that the m7G cap-binding protein eIF4E and one of its interaction partners, eIF4E-Transporter, are also components of mammalian P bodies. We further show, by FRET, that they form a molecular complex with each other in P bodies in vivo. Additionally, eIF4E interacts with the translational repressor rck/p54. In contrast, other translation initiation factors such as eIF4G or components of the translation machinery were not detected in these cytoplasmic foci, ruling out the possibility that P bodies are sites where active translation can occur. It was possible to show that eIF4E requires that it is bound to the m7G cap of the mRNA and interact with eIF4E-T in order to be targeted to the P bodies. Altogether, eIF4E-T prevents the formation of a translationally active mRNP by interacting with eIF4E and thus plays a role in remodeling events that render mRNAs available for degradation. No particular sequence within eIF4E-T was found sufficient for its localization to the P bodies, but only the full-length eIF4E-T molecule could assemble inside these bodies. Using RNAi-mediated knockdowns we observed that a subset of P body factors, including eIF4E-T, LSm1, rck/p54, and Ccr4 are required for the accumulation of each other and of eIF4E in P bodies. Furthermore, cycloheximide treatment of cells lead to disassembly of the entire structure, indicating that the accumulation of LSm1, eIF4E, eIF4E-T, and rck/p54 in P bodies also requires that an mRNA flux be available. These results suggest that these factors are targeted to P bodies as part of a larger mRNP complex and that they are essential for P body formation and function in mRNA processing. A block in the decay of the mRNA body upon depletion of the decapping enzyme Dcp2 or the exoribonuclease Xrn1, leads to an increase in the size and number of P bodies. A distinct hierarchy of remodeling steps and sequence of events can be inferred towards formation of the P bodies whereas early acting factors are represented by the deadenylase Ccr4, LSm1, eIF4E-T and rck/p54 and late joining factors include Dcp2 and Xrn1. Mass spectrometric analyses helped to identify a novel RNA-binding protein residing in the P bodies, which has preference for Cytosine-rich elements and hints to a mechanism implying sequence-specific silencing of mRNAs inside the P bodies.