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. 2001 Apr 24;98(9):4961-5.
doi: 10.1073/pnas.091100698.

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

G Mayer  1 , M Blind, W Nagel, T Böhm, T Knorr, C L Jackson, W Kolanus, M Famulok
Affiliations

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

G Mayer et al. Proc Natl Acad Sci U S A. .

Abstract

ADP-ribosylation factor (ARF) GTPases and their regulatory proteins have been implicated in the control of diverse biological functions. Two main classes of positive regulatory elements for ARF have been discovered so far: the 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 fungal metabolite brefeldin A, has enabled documentation of the involvement of 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 recognize the Sec7 domain of cytohesin 1. Some aptamers inhibit guanine-nucleotide exchange on ARF1, thereby preventing ARF activation in vitro. Among them, aptamer M69 exhibited unexpected specificity for the small GEFs, because it does not interact with or inhibit the GEF activity of the related Gea2-Sec7 domain, a member of the class of large GEFs. The inhibitory effect demonstrated in vitro clearly is observed as well in vivo, based on the finding that M69 produces similar results as a dominant-negative, GEF-deficient mutant of cytohesin 1: when expressed in the cytoplasm of T-cells, M69 reduces stimulated adhesion to intercellular adhesion molecule-1 and results in a dramatic reorganization of F-actin distribution. These highly specific cellular effects suggest that the ARF-GEF activity of cytohesin 1 plays an important role in cytoskeletal remodeling events of lymphoid cells.

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Figures

Figure 1
Figure 1
Sequences of nucleic acids and proteins. (A) Nucleic acid sequences of the RNA aptamers M5, M56, M69, TR-M69, and TR-ML1, respectively. Black, initial random sequence; blue, 5′ and 3′ terminal regions for primer annealing; yellow, 5′ and 3′ stem-loop sequences of the TR-expression cassette. (B) Secondary structure prediction of M69 proposed by the program mfold (26). Blue, constant 5′ and 3′ terminal regions of the RNA aptamer. (C) Amino acid sequence of cytohesin 1. Red, C1-Sec7 domain, responsible for M69 binding; green, amino acid E157 residue critical for ARF-GEF activity mutated in cytohesin 1(E157K).
Figure 2
Figure 2
Inhibition of GDP/GTP exchange on ARF-1 by M69. (A) GDP/GTP-exchange activity of cytohesin 1, C1-Sec7, C2-Sec7, and Gea2-Sec7 on ARF-1 in the presence of M69 (solid bars) and unselected RNA library (shaded bars). The amount of bound [γ35-S]GTP after 30 min without RNA was defined as 100%. (B) Concentration dependence of inhibition of cytohesin 1-catalyzed GDP/GTP exchange on ARF-1 by M69 (■) and unselected pool (♦). (C) Time course of inhibition of cytohesin 1-catalyzed GDP/GTP exchange on ARF-1 by M69 (▴) vs. noninhibited negative control (■). (D) GDP/GTP-exchange activity of cytohesin 1, C1-Sec7, C2-Sec7, and Gea2-Sec7 on ARF-1 in the presence of BFA.
Figure 3
Figure 3
Adhesion of nonstimulated (shaded bars) and PMA-stimulated (solid bars) Jurkat E6 cells to ICAM-1 as a function of aptamer expression. vT7, single infection with vaccinia viruses expressing T7-RNA polymerase; vTR-M69, single infection with vTR-M69; vT7/vTR-ML1, double infection with vT7 and the nonbinding negative control sequence TR-ML1; vT7/vTR-M69, double infection with vaccinia viruses expressing T7-RNA polymerase (vT7) and TR-M69.
Figure 4
Figure 4
Actin cytoskeleton staining visualized by fluorescence (A–D) and confocal microscopy (E–H). (A) Single infection with vT7. (B) Single infection with vTR-M69. (C) Double infection with vT7 and vTR-ML1. (D) Double infection with vT7 and vTR-M69. Shown is actin cytoskeleton staining after expression of the constant regions of human IgG antibody CH1 and CH2 (Ig) (E), the Ig/cytohesin 1 fusion protein (F), the cytohesin 1(E157K) mutant/Ig fusion protein (G), and the TR-M69 intramer (H).
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