An intronic regulatory element that overlaps with U1ex13-11
binding site regulates exon 13 splicing and forms a stem-loop RNA
secondary structure
Among the CFTR exons evaluated, exon 13 was the most challenging to
rescue. In fact, at variance to the other exons, we have identified only
one ExSpeU1, U1ex13-11, with a reliable splicing rescue activity on both
1863 C>T and 1898+3A>G mutations (Fig. 1A).
Thus, we decided to explore if there is any specific reason for this
apparent lack of efficiency. To this aim we focussed on intronic
regulatory elements within the ExSpeU1 binding region performing
consecutive intronic deletions in WT, 1863C>T and
1898+3A>G (Fig. 3A) minigenes. Interestingly, deletion of
bases in position 12-15 and 17-23 downstream of the 5’ss, but not in the
other positions, improved splicing in both the WT and in the
1863C>T contexts (Fig. 3A, lanes 2,3 and 9,10,
respectively). These deletions, however, did not affect splicing in the
1898+3A>G minigene. These results indicate that the binding
sequence of the active U1ex13-11 (between positions +11 and +26)
overlaps with an Intronic Splicing Silencer (ISS) located between
position +11 and +23. In this context, the strength of the ISS is not
sufficient to recover exon 13 skipping caused by the 5’ss mutation. In
addition, analysis with RNA-mfold (Wang et al., 2017) showed that the
ISS is located within a 7bp-long stem-loop that include the 5’ss (Fig.
3B). This configuration suggests that the peculiar RNA secondary
structure where the ISS is located might limit the accessibility of the
U1snRNP to the 5’ss, as previously reported in other cases (Buratti &
Baralle, 2004). To clarify the ISS mechanism in detail we performed site
directed mutagenesis experiments assisted by RNA secondary structure
analysis in the loop (positions +12 and +13), in the stem (positions +14
to +19) and outside the structure (positions +20 to +23). The resulting
effect on splicing expressed as changes in percentage of exon inclusion
was then related to the predicted effect on the secondary structure
expressed as ΔG, that is the quantity of energy needed to fully break
the secondary structure (Fig. 3D and Table S1). Mutagenesis from the +14
to +19 position are predicted to open the stem of the structure and
accordingly in splicing assay they increase exon inclusion (Fig. 3C,
lanes 8-18). In contrast, the mutants predicted to close the loop,
namely +13C, +12C13C and +12A13C, had a negative effect on exon 13
inclusion and induced nearly complete skipping (Fig. 3C, lanes 5, 25,
26). All other mutants in the loop (+12T, +12A, +12C, +13T, +13G
+12A13T, +12C13G) and those outside of the structure (+20A, +20G, +21G,
+22T, +22A, +23G), that do not significantly affect the secondary
structure, had no significant effect on splicing. Based on the predicted
changes in ΔG (Table S1) and the resulting change in percentage of exon
inclusion we modelled association between the change in stability of the
secondary structure and the impact on splicing (Fig. 3D). This analysis
showed a clear sigmoidal association between splicing of exon 13 and ΔG,
supporting the hypothesis that the stem-loop structure at CFTR exon 13
5’ss is involved in splicing regulation and that this might restrict the
effect of ExSpeU1s to specific intronic binding sites.