Fig. 7

The triloop conformation of TSL2 facilitates SMN2 E7 splicing. a Sequence of non-mutated (n.m.) TSL2 or TSL2 carrying the 2A, 2C, or 3G17G mutations¹⁶. b Graph recapitulating that mutation 2 A increases E7 splicing and PK4C9 activity (expressed in percentage, %) in HeLa cells transfected with SMN2^E6−to−8 minigenes; whereas 2C, and 3G17C decrease them (see also Fig. 5b, c). MIP maximum E7 increment possible (see Eq. 2). c Representative structures of the 2A, 2C and 3G17C RNAs taken from their 40-ns MD trajectories. 2A TSL2 exists mainly as a triloop, whereas 2C and 3G17C TSL2 exist mainly as pentaloops. d Quantification of the distance between the C1’ atoms of residues A1 and U19 throughout the MD trajectories, as an indicator of terminal TSL2 opening. The 2A structure shows the largest opening, whereas the 3G17G structure shows the shortest one. e Per-residue comparison of the mobility of the 2A, 2C and 3G17C mutant structures (RMSF). The mobility of loop residues A8–U11 is the highest in the 3G17C structure and lowest in the 2A mutant. f RT-PCR from HeLa cells transfected with an SMN2^E6−to−8 minigene carrying either n.m. TSL2 or the 8G12C double mutation. The 8G12C mutation, which stabilises TSL2 in its triloop conformation, increased E7 splicing to nearly 100% (n = 3 biological replicates). g, h Schematic model of the proposed mechanism of action of PK4C9. Triloop and pentaloop conformations of TSL2 coexist, with triloop conformations displaying better access of the 5′ ss and E7 splicing. PK4C9 would bind to pentaloop TSL2 directly improving 5′ ss exposure and indirectly causing a shift to the triloop (h). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. p-values were obtained by applying non-paired, two-tailed t tests with Welch corrections. The graph shows mean values ± SEM