Analysis of MicroRNA-mediated translation activation of in vitro transcribed reporters in quiescent cells

Abstract

Quiescence (G0) is defined as an assortment of cell cycle arrested states that exhibit distinct properties. Leukemias harbor a subpopulation of G0 cells that can be enriched by growth factor deprivation or serum starvation. Target site reporters with shortened poly(A) tails show translation activation by microRNAs, via a noncanonical mechanism, when introduced into the nucleus of G0 cells. This is because recruitment by the activation causing FXR1a-microRNA-protein complex (FXR1a-microRNP) is nuclear and requires shortened poly(A) tails to avoid repressive factors and canonical translation. When introduced into the cytoplasm, target mRNAs and microRNAs are directed toward repression rather than translation activation. Leukemic cell lines are difficult to transfect but can be routinely nucleofected—where in vitro transcribed mRNA reporters and microRNAs are introduced into the nucleus of G0 leukemic cells. Nucleofection of a microRNA target reporter and either cognate, targeting microRNA, or control microRNA, into the nucleus of G0 cells, enables analysis of translation activation by microRNAs in G0. We discuss a modified protocol that we developed for transfection of mRNAs along with microRNAs to test translation regulation by microRNAs in G0 leukemic cells.