mRNA Targeting to Endoplasmic Reticulum Precedes Ago Protein Interaction and MicroRNA (miRNA)-mediated Translation Repression in Mammalian Cells *

摘要

Background: miRNA, Ago2, and target messages are enriched on ER in metazoan cells. Results: Polysome association of newly formed mRNA precedes Ago2 binding on ER membrane and its translation repression. Conclusion: miRNA repression process is compartmentalized in mammalian cells, and polysome association of mRNA happens before the repression. Significance: This work provides a detailed understanding of the miRNA-mediated gene repression and mRNA compartmentalization in mammalian cells. MicroRNA (miRNA) binds to the 3′-UTR of its target mRNAs to repress protein synthesis. Extensive research was done to understand the mechanism of miRNA-mediated repression in animal cells. Considering the progress in understanding the mechanism, information about the subcellular sites of miRNA-mediated repression is surprisingly limited. In this study, using an inducible expression system for an miRNA target message, we have delineated how a target mRNA passes through polysome association and Ago2 interaction steps on rough endoplasmic reticulum (ER) before the miRNA-mediated repression sets in. From this study, de novo formed target mRNA localization to the ER-bound polysomes manifested as the earliest event, which is followed by Ago2 micro-ribonucleoprotein binding, and translation repression of target message. Compartmentalization of this process to rough ER membrane ensures enrichment of miRNA-targeted messages and micro-ribonucleoprotein components on ER upon reaching a steady state.