Controlling small guanine-nucleotide-exchange factor function through cytoplasmic RNA intramers

ADP-ribosylation factor (ARF) GTPases and their regulatory proteins have be en implicated in the control of diverse biological functions. Two main clas ses of positive regulatory elements for ARF have been discovered so far: th e large Sec7/Gea and the small cytohesin/ARNO families, respectively. These proteins harbor guanine-nucleotide-exchange factor (GEF) activity exerted by the common Sec7 domain. The availability of a specific inhibitor, the fu ngal metabolite brefeldin A, has enabled documentation of the involvement o f the large GEFs in vesicle transport. However, because of the lack of such tools, the biological roles of the small GEFs have remained controversial. Here, we have selected a series of RNA aptamers that specifically recogniz e the Sec7 domain of cytohesin 1. Some aptamers inhibit guanine-nucleotide exchange on ARF1, thereby preventing ARF activation in vitro. Among them, a ptamer M69 exhibited unexpected specificity for the small GEFs, because it does not interact with or inhibit the CEF activity of the related Gea2-Sec7 domain, a member of the class of large GEFs, The inhibitory effect demonst rated in vitro clearly is observed as well in vivo, based on the finding th at M69 produces similar results as a dominant-negative, GEF-deficient mutan t of cytohesin 1: when expressed in the cytoplasm of T-cells, M69 reduces s timulated adhesion to intercellular adhesion molecule-1 and results in a dr amatic reorganization of F-actin distribution. These highly specific cellul ar effects suggest that the ARF-GEF activity of cytohesin 1 plays an import ant role in cytoskeletal remodeling events of lymphoid cells.