Figure 3

G4 Structures Regulate CYP3A5 Expression and Represent Novel Targets for ASO Modulation. (a) Schematic diagram of the CYP3A5 gene showing the location of a putative G4 motif in Intron 3. Cation-specific stabilization of native G4 structure shifts the splice acceptor back to the reference exon 4 splice acceptor, creating an in frame CYP3A5*3/*3 transcript that bypasses pseudo-exon introduction and suppression of wild-type protein translation via PTC or NMD-related mechanisms. Salt-sensitive splice correction of *3 transcripts (shown in Fig. 2a) can be abrogated using the G4 disrupting PMO (Table 1). (b) Computational analysis of the CYP3A5 gene (see Supplemental Fig. 2) and RNA/DNA gel shift assays (shown here), confirmed the existence of a G4-like structure in the CYP3A5 intron 3 region. Control lanes 1 and 2 depict the mobility of the 151 nucleobase, CYP3A5 intron 3 “C-strand” DNA, in KCl-enriched running buffer [100 mM KCl]. The C-strand does not display faster migration typical of a G4 structure in the presence of KCL, and a G4 disrupting PMO does not alter C-strand mobility (lane 2). In contrast, the 151 nucleobase “G-strand” DNA demonstrated enhanced mobility bands reflecting both inter- and intra-strand G4 formation in KCl-enriched running buffer (lane 3). Addition of the G4 disrupting PMO diminishes the enhanced mobility bands at low concentration (1:1 molar ratio; lane 4) and ablates the enhanced mobility bands at high concentration (5:1 molar ratio; lane 5). (c) A 151 nucleobase RNA strand also forms a putative G4-structure in KCl-enriched running buffer (Lane 1). Addition of increasing amounts of G4 disrupting PMO to the G4 RNA (Lane 2 – 1:1 PMO:RNA ratio, Lane 3 – 3:1 PMO:RNA ratio, and Lane 4 – 5:1 PMO:RNA ratio) shifts the RNA structure to the slower mobility form. (d) qPCR analysis of CYP3A5 mRNA from HEK293 cells exposed to no PMO and normal media (HEK control), scramble PMO (1 µM, 48 h.) and KCl (12 mM) (HEK + Scr + KCl), G4 disrupting PMO only (1 µM, 48 h.) (HEK + G4 Disrupt), and G4 disrupting PMO (1 µM, 48 h.) and KCl (12 mM) (HEK + G4 Disrupt + KCl). Significant statistical differences were monitored by ANOVA; p value < 0.01. Post test p < 0.01 = **. (e) (Top) A schematic illustrating the location of the EJC in relation to the cryptic splice acceptor in intron 3 and the G4 structure. PMOs designed to skip exon 4 in CYP3A5 are also shown (-17 = E4SA-17, -9 = E4SA-9, -3 = E4SA-3, and + 94 = E4SA + 94). (Bottom) Endpoint PCR results for RNA expression in HEK293 cells treated with three PMOs (E4SA-3, E4SA-9, or a scramble control (Scr); 1 µM; 48 h.) and 100 mM NaCl are shown. The Scr. oligomer did not induce stable transcripts, while exon 4 skipping PMOs (E4SA -3 and -9) induced transcripts that both include and exclude exon 4. The addition of high salt media diminished the cell’s ability to express transcripts including exon 4. Cationic-specific stabilization of the transcript may have improved the ability of exon skipping PMOs to properly bind the transcript near the EJC assembly zone and promote targeted exon 4 exclusion. Molecular drawings were generated using The PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC.