Genome-wide analysis of Staufen1-bound mRNAs in prometaphase

The goal of this experiment was to identify Staufen1-bound mRNAs in prometaphase cells. Stau1 abundance fluctuates through the cell cycle: it is high in the G2 phase of the cell cycle and rapidly decreases as cells transit through mitosis. The importance of controlling Stau1 levels was underscored by the observation that its overexpression impairs mitosis as well as proliferation of transformed cell lines. As a major post-transcriptional regulator, Stau1 may exert its role(s) through the spatial and/or temporal regulation of its bound mRNAs. Therefore, to gather clues to support this possibility, we identified Stau1-bound mRNAs in prometaphase as a means to identify mRNAs that could be subjected to post-transcriptional modulation when Stau1 is partly degraded in mitosis. HEK293T cells were transfected with plasmids coding for Stau155-FLAG or the empty vector as control. Cells were synchronized in prometaphase with nocodazole. Stau1-bound mRNAs were isolated after immunoprecipitation using anti-FLAG antibody.