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

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• ADP-ribosylation factor (ARF) GTPases regulate vital cellular functions.
• Two classes of ARF regulators exist: large Sec7/Gea and small cytohesin/ARNO families, both possessing guanine-nucleotide-exchange factor (GEF) activity via a Sec7 domain.
• While large GEFs are well-studied using inhibitors like brefeldin A, the roles of small GEFs remain less understood due to a lack of specific tools.

Purpose of the Study:

• To develop specific inhibitors for small ARF-GEFs.
• To investigate the biological functions of the small GEF, cytohesin 1.
• To elucidate the role of cytohesin 1 in cellular processes like T-cell adhesion and cytoskeletal dynamics.

Main Methods:

• Selection of RNA aptamers targeting the Sec7 domain of cytohesin 1.
• In vitro assays to assess aptamer inhibition of guanine-nucleotide exchange on ARF1.
• In vivo studies involving expression of aptamer M69 in T-cells to observe cellular effects.

Main Results:

• Aptamer M69 specifically recognizes and inhibits the GEF activity of cytohesin 1.
• M69 does not inhibit the related large GEF, Gea2-Sec7 domain, demonstrating high specificity.
• In T-cells, M69 expression reduced stimulated adhesion to ICAM-1 and caused significant F-actin reorganization.

Conclusions:

• The RNA aptamer M69 serves as a specific inhibitor for the small ARF-GEF, cytohesin 1.
• The study provides in vitro and in vivo evidence for M69's inhibitory effects.
• Cytohesin 1's ARF-GEF activity is crucial for cytoskeletal remodeling in lymphoid cells, particularly in adhesion and actin dynamics.