Precise Tools for Site-Directed RNA Editing and Induction of NO Signaling

Abstract

RNA editing represents an important class of posttranscriptional alterations with various physiological and pathophysiological effects. ADARs catalyze deaminations of adenosines in double stranded RNA to inosines (A-to-I), APOBECs deaminations of cytidines to uridines (C-to-U). The SNAP-ADAR system provides a tool for site-directed A-to-I RNA editing by fusion of a catalytically active ADAR deaminase domain to the self-labeling SNAP-tag. SNAP-tag reacts covalently with short guideRNAs carrying an O6-benzylguanine modification, thus allowing for targeted recruitment of SNAP-ADAR to a specific adenosine by Watson Crick base pairing. In the dissertation at hand, the SNAP-ADAR platform has now been expanded by HALO-ADAR as second editase with orthogonal recruitment mechanism. While SNAP-ADAR is steered with O6-benzylguanine modified guideRNAs, chloroalkyl modifications were applied for HALO-ADAR. This permitted the independent, parallel steering of SNAP-ADAR2 and HALO-ADAR1 in mammalian cells, which yielded optimal editing for an extended substrate scope. Moreover, the combination of HALO-ADAR1 with APOBEC1-SNAP enabled targeted, concurrent and orthogonal A-to-I and C-to-U editing, which may be exploited for the investigation of the interplay between A-to-I and C-to-U editing events in the future.

Furthermore, SNAP-ADAR editing was put under control of small molecule induction. Design of separate SNAP-GID1A and GAI1-92-ADAR1 fusions rendered the recruitment of editing activity dependent on chemically induced dimerization of GID1A and GAI1-92 with gibberellic acid (GA3). As a result, tightly controlled GA3 inducible A-to-I editing was achieved. The extent of editing was tunable by GA3 dosage control, which is particularly beneficial for sites requiring careful adjustment to exclude potential detrimental effects.

Additionally, a tool for photoinduced activation of the NO-cGMP signaling pathway has been developed. Nitric oxide (NO) represents a versatile secondary messenger. Among others, it induces cGMP production and consequently the cGMP signaling cascade with various implications in smooth muscle tone regulation as well as neuronal processes. The highly reactive NO can be supplied by NO releasing drugs, including diazeniumdiolates, in physiological settings. In the work at hand, the N-bound diazeniumdiolate of pyrrolidine, PYRRO/NO, which releases NO within seconds under physiological conditions, has been photoprotected as MeNPOM-PYRRO/NO in order to stabilize the compound in the absence of light. Application of MeNPOM-PYRRO/NO in primary vascular smooth muscle cells allowed for generation of well-defined cGMP signals upon illumination with long-wavelength UV light. The excellent spatiotemporal control provided by photoactivation should enable spatial control at subcellular level, which may prove valuable for the prospective elucidation of cGMP compartmentalization.