Mitogen-responsive expression of RhoB is regulated by RNA stability

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Abstract
The small GTPase-encoding gene RhoB is strongly induced as part of the immediate early response of serum-stimulated fibroblasts. In this report, we have characterized the mechanism for growth factor responsiveness of RhoB in Rat-2 fibroblasts. By Northern blotting and ribonuclease protection, we observed low or barely detectable levels of RhoB mRNA in quiescent cells, but expression was transiently induced in response to serum stimulation, such that the mRNA peaked within 30 min and then declined over the next hour. Analysis of the rat promoter revealed cis-elements conserved with the mouse and human genes, including a pair of CEBP sites near the transcriptional start site. However, in contrast to the analysis of RNA, RhoB promoter fusions were constitutively expressed in quiescent cells in transient transfections, and were unaffected by serum. Similarly, stable RhoB promoter integrants were highly expressed in quiescent cells, and growth factor caused a slight decrease in activity. This indicates that growth factor-inducible RhoB expression cannot be mediated by transcriptional activation. We then examined decay of the RhoB mRNA and found that serum caused significant stabilization. Additionally, fusion of the 3' RhoB untranslated region (UTR) to a constitutively expressed reporter gene caused serum and growth factor as well as DNA damage-inducible expression. These observations are consistent with the view that RhoB mRNA is produced constitutively but its abundance is controlled in response to growth factors, and other signals including DNA damage, by stabilization through elements within the 3' UTR.