![]() ![]() The adenosine-to-inosine (A-to-I) editing reaction of RNA is performed by dsRNA-specific adenosine deaminases (ADARs), which convert adenosine to inosine via hydrolytic deamination. Imino protons of the central G-quartet remain (and often 1–2 additional guanine imino signals from terminal G-quartets) (34) after 1 h of sample incubation in 100% D 2O (see also Figure S6). Underlined G in the sG3 wt sequence indicates site of G-to-I substitution. sG3 wt (5′-AGGGCUGGGCUG GGCGGGA-3′) refers to a truncated version of G3 wt where one G from each of the two GGGG-tracts was removed to stop G-register exchange dynamics (33) during NMR measurements. (j–m) 1H NMR spectra of the imino region between 10 and 12 ppm in the presence of 25 mM KCl corresponding to sG3 wt in (j) 10% D 2O, (k) sG3 I in 10% D 2O, (l) sG3 wt in 100% D 2O, and (m) sG3 I in 100% D 2O. (h, i) Suggested mode of quadruplex unfolding: (h) model for G3 wt and I 17 and (i) model for I 16 and I 18. The curves indicate quantified stoichiometries as a function of temperature after subtracting the nonspecific adduct contributions. (d–g) Thermal melting experiment monitored with mass spectrometry at 0.1 mM KCl for: (d) G3 wt, (e) I 17, (f) I 16, and (g) I 18. (29) Inset: Chemical structure of 7-deazaguanosine. Incorporation of 7-deazaguanosine at position 17 abolishes quadruplex formation. (c) Melting curves at 3 mM KCl for I, A, or 7-deaza G substitutions at position 17 normalized to the signal at 25 ☌. Data represent mean + SD from three consecutive scans. (b) Melting temperatures at 295 nm in 3 mM KCl for G3 wt (gray) and substitution variants: G-A (blue) and G-I (red). Annotated in red (16–17–18) is G-tract where guanosines were substituted to inosines or adenosines. Structure of a conventional RNA G3-quadruplex (rG4) model sequence (G3 wt 5′-AAAAGGGCUGGGGCUG 16G 17G 18CGGGGA-3′) with three quartets linked by loops with parallel orientation. (a) Inosine lacks the 2-amino group, leading to the loss of one hydrogen bond with the N 7 of the neighboring guanosine in a G 3I-quartet. Biophysical characterization of GI-quadruplex formation in vitro. (8,9) These structures may exhibit reduced stabilities and, hence, lowered barriers for conformational exchanges that facilitate functional responses. (2) Moreover, it is likely that many unusual rG4s exist that cannot yet be predicted by current computational methods. (8−10) For example, transcriptome-wide searches have identified numerous putative rG4s, comprising G2-tracts and long, structured loops. (7) However, noncanonical rG4s with highly unusual structures are increasingly described. A canonical three-stacked rG4 typically has short-loops and a stable compact structure. (4,5) A G-quadruplex is stabilized by eight hydrogen bonds (H-bonds) between the Watson–Crick/Hoogsten faces of guanines, by π–π stacking between the planar faces of the G-quartets and by the coordination of potassium ions with guanine oxygens in the central cavities (6) ( Figure 1a). (3) They consist of stacked G-quartets, formed from sequences comprising four tracts of guanosine repeats. G-quadruplexes are widespread in the transcriptome (1,2) where they influence transcription termination, polyadenylation, splicing, and translation and serve as binding sites for RNA-binding proteins. In summary, RNA GI-quadruplexes are a previously unrecognized structural motif that may contribute to the regulation of gene expression in vivo. To study inosine-induced structural changes in a naturally occurring RNA, we use a synthetic approach that enables site-specific inosine incorporation in long RNAs. They exhibit moderately reduced thermal stability compared to conventional G-quadruplexes. GI-quadruplexes adopt parallel topologies, stabilized by potassium ions. We demonstrate the formation of inosine-containing rG4s (GI-quadruplexes) in vitro and verify their activity in cells. Here, we provide biophysical, chemical, and biological evidence that A-to-I exchange can activate latent rG4s by filling incomplete G-quartets with inosine. Adenosine-to-inosine (A-to-I) editing is a common chemical modification of RNA which introduces a nucleobase that is iso-structural with guanine, thereby changing RNA base-pairing properties. For instance, stem-loops and G-quadruplexes (rG4s) are dynamic motifs in mRNAs that influence gene expression. It is well-accepted that gene expression is heavily influenced by RNA structure. ![]()
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