Mouse Pxt1 expression is regulated by Mir6996 miRNA

Mouse Pxt1 gene is expressed exclusively in male germ cells and encodes for a small, cell death inducing protein. However, upon PXT1 interaction with BAG6, cell death is prevented. In transiently transfected cell lines the PXT1 expression triggered massive cell death, thus we ask the question whether the interaction of PXT1 and BAG6 is the only mechanism preventing normal, developing male germ cells from being killed by PXT1. The Pxt1 gene contains a long 3'UTR thus we have hypothesized that Pxt1 can be regulated by miRNA. We have applied Pxt1 knockout and used Pxt1 transgenic mice that overexpressed this gene to shed more light on Pxt1 regulation. Using the ELISA assay we have demonstrated that PXT1 protein is expressed in adult mouse testis, though at low abundance. The application of dual-Glo luciferase assay and the 3'UTR cloned into p-MIR-Glo plasmid showed that Pxt1 is regulated by miRNA. Combining the use of mirDB and the site-directed mutagenesis further demonstrated that Pxt1 translation is suppressed by Mir6996-3p. Considering previous reports and our current results we propose a model for Pxt1 regulation in the mouse male germ cells.