Figure 8.
(A) Shows a diagrammatic representation of several positive elements that regulate SMN1/SMN2 exon 7 inclusion. The elements analyzed in this experiment are the first nucleotide of exon 7 (1G), the Tra2β-binding site (Tra2), the conserved tract in the middle of the exon (ConsTract) and the intronic splicing enhancer (element-2) in IVS7. Also shown is the dominant ISS element (ISS-N1). The graphical representation underneath shows the levels of exon 7 inclusion following mutation of all of these elements in a suitable minigene construct. Also included are the basal exon 7 inclusion levels from SMN2 exon 7 that carries a natural C6U substitution. (B) Shows the effect of the same mutations/substitutions when the ISS-N1 sequence has been deleted from the minigene constructs. An analogous situation which regards the CFTR exon 9 sequence is schematically represented in (C) in which the (UG)m(U)n repeated motif near the 3′ss of this exon has been shown to bind a powerful negative regulator of splicing, TDP-43. Several other cis-acting elements are also shown in this diagram: a CERES element within the exon, a positive polypyrimidine sequence near the 5′ss (PCE) that binds TIA-1, and a human-specific ISS in IVS9 that binds members of the SR protein family. The graph below shows that mutations within these elements are strongly inhibitory with respect to the basal inclusion level on a TG11T5 background (lane CFTR exon 9). Nonetheless, also in this case the removal of TDP-43 using siRNA technology rescues the effect of all these negative mutations (D).