MicroRNA-mediated mRNA Translation Activation in Quiescent Cells and Oocytes Involves Recruitment of a Nuclear microRNP

Abstract

MicroRNAs can promote translation of specific mRNAs in quiescent (G0) mammalian cells and immature Xenopus laevis oocytes. We report that microRNA-mediated upregulation of target mRNAs in oocytes is dependent on nuclear entry of the microRNA; cytoplasmically-injected microRNA repress target mRNAs. Components of the activation microRNP, AGO, FXR1 (FXR1-iso-a) and miR16 are present in the nucleus and cytoplasm. Importantly, microRNA target mRNAs for upregulation, Myt1, TNFα and a reporter bearing the TNFα AU-rich, microRNA target sequence, are associated with AGO in immature oocyte nuclei and AGO2 in G0 human nuclei, respectively. mRNAs that are repressed or lack target sites are not associated with nuclear AGO. Crosslinking-coupled immunopurification revealed greater association of AGO2 with FXR1 in the nucleus compared to cytoplasm. Consistently, overexpression of FXR1-iso-a rescues activation of cytoplasmically-injected RNAs and in low density, proliferating cells. These data indicate the importance of a compartmentalized AGO2-FXR1-iso-a complex for selective recruitment for microRNA-mediated upregulation.

FXR1a-associated microRNP: A driver of specialized non-canonical translation in quiescent conditions

“…29 In proliferating cells, FXR1a overexpression leads to microRNA mediated translation activation. 75 Consistently, proteomic studies in FXR1 depleted cells revealed decrease in protein levels of certain mRNAs that are targets of activation and require FXR1 for their polysome association. 31 Third, FXR1 does not interact with GW182 and does not lead to repression but instead promotes activation of translation.…”

“…25,31,75 Several intriguing questions emerge from these studies and remain to be addressed. First, it is not known how FXR1a-microRNP selects its targets for activation in G0.…”

“…Consistently, we find that Cyclin E mRNA that is not recruited by FXR1a-microRNP in the nucleus, is repressed in G0. 31,75,120 How specific microRNAs are recruited to this complex remain to be ascertained. Fifth, mRNAs recruited by the FXR1a-microRNP need to have short poly(A) tails to avoid PABP and thereby, avoid repression by GW182 and canonical translation that is inhibited in these conditions.…”

“…75 FXR1a-microRNP activates translation of reporter mRNAs bearing microRNA target sites in the 3 0 UTR, and of specific endogenous mRNAs like TNFa and MYT1 in G0 mammalian cells and Xenopus immature oocytes respectively. 17,25,31 Proliferating THP1 cells, treated with mTOR inhibitor Torin1 that blocks 4EBP phosphorylation and thereby, canonical translation-recreating conditions similar to G0 cells-show microRNA mediated translation activation of reporter mRNAs.…”

“…29,31 In G0 and distinct oocyte stages, 115,116 the association of repressor GW182 protein with AGO2 may be reduced, 114,117-119 which may permit activation of specific mRNAs. Fourth, in order to get translation activation, target mRNAs and the microRNAs in quiescent conditions have to be recruited in the nucleus 75 by AGO2 and FXR1. mRNAs not associated in the nucleus with the FXR1a-microRNP are not translationally upregulated, and could be subject to repression by the canonical microRNP.…”