ROCK inhibition enhances microRNA function by promoting deadenylation of targeted mRNAs via increasing PAIP2 expression.
Bottom Line: Here, we report that global miRNA function can be enhanced by Rho-associated, coiled-coil-containing protein kinase (ROCK) inhibitors.In the presence of a ROCK inhibitor, PAIP2 expression is enhanced by the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) through increased ROCK1 nuclear localization and enhanced ROCK1 association with HNF4A.Our data reveal an unexpected role of ROCK1 as a cofactor of HNF4A in enhancing PAIP2 transcription.
Affiliation: Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.Show MeSH
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Mentions: Both phosphorylation and lysine acetylation of HNF4A affect its transcriptional activity (31–33). Therefore, we first examined changes in post-translational modifications of HNF4A after ROCK inhibition, but detected no changes in such modifications under our experimental conditions (Supplementary Figure S6A). Next, to determine the mechanism by which the transcriptional ability of HNF4A is enhanced by ROCK inhibition, we investigated the interaction between HNF4A and two ROCK paralogs by immunoprecipitation of overexpressing flag-tagged HNF4A. There was a clear interaction between HNF4A and cellular endogenous ROCK1 and not ROCK2 (Figure 5A). Moreover, the interaction between HNF4A and ROCK1 was significantly enhanced upon treatment with the ROCK inhibitor (Figure 5A). To confirm the binding between HNF4A and ROCK1, we used HCT116 cells transfected with a halo-tagged ROCK1-expressing plasmid and flag-tagged HNF4A-expressing plasmid. Endogenous ROCK1 and halo-tagged ROCK1 proteins were co-precipitated by immunoprecipitation of the flag-tagged HNF4A protein. Consistent with the data shown in Figure 5A, the interaction between HNF4A and ROCK1 was enhanced upon treatment with the ROCK inhibitor (Figure 5B).
Affiliation: Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.