Identification of G-quadruplexes in long functional RNAs using 7-deazaguanine RNA

Abstract

RNA G-quadruplex (G4) structures are thought to affect biological processes, including translation and pre-mRNA splicing, but it is not possible at present to demonstrate that they form naturally at specific sequences in long functional RNA molecules. We developed a new strategy, footprinting of long 7-deazaguanine-substituted RNAs (FOLDeR), that allows the formation of G4s to be confirmed in long RNAs and under functional conditions.

Figure 1. The functional Bcl-x-681 RNA contains G4s in vitro

(a) Schematic representation of native Bcl-x (top) and Bcl-x-681 transcript (bottom) used in this study. The size of introns and exons are indicated in nucleotides below the diagrams. X_L and X_S 5’ splice sites are indicated above the diagrams. (b) In vitro splicing assay of Bcl-x-681. Bands corresponding to the unspliced transcript, the X_L and the X_S products are labelled. The full-length gel is displayed in Supplementary Fig. 11 (c) Electrophoretic-mobility shift assay of native (left) and 7-deaza-G substituted (right) Bcl-x-681 in the absence or presence of increasing amount of BG4 antibody (0, 150 pM, 1.5 nM, 15 nM, 150 nM and 1.5 μM). The full-length gel is displayed in Supplementary Fig. 11. (d) Mapping of RNA footprinting of Bcl-x-681. Nucleotides having a different footprinting pattern upon 7-deaza-GTP substitution are circled and are located mainly to the X_S and X_L domains. Putative G4s are labelled Q1 to Q6.

Figure 2. The Bcl-x-681 RNA contains G4s in functional conditions

(a) Schematic drawing of the RNAse H cleavage of native Bcl-x-681 on the secondary structure model of Bcl-x-681. Accessible regions (average >60% cleavage) are in black, and protected regions (average <40% cleavage) are in grey. RNAse cleavage experiments were done in triplicate and are presented in supplementary Figure 9A. (b) Significance of changes in RNAse H cleavage between 7-deaza-G and native RNA in nuclear extract. Three experiments were done on each transcript and, for each oligonucleotide, a Student’s t test was done to establish the probability that the two sets of results might come from the same population. Values of p below 0.05 indicate that the cleavage of the two transcripts was significantly affected by deaza substitution. Representative experiments on the two transcripts are shown in Supplementary Figure 10.