In silico analysis predicts aberrant splicing events of NOTCH1 pre-mRNA. (A) Schematic representation of C-terminal NOTCH1 pre-mRNA. NOTCH1 wt mRNA with the PEST domain coding sequence in exon 34 is spliced canonically (left), while upon mutation alternative splicing is induced (right). Here, the mutated 3′UTR sequences as indicated create acceptor sites, which can form a stronger acceptor motif with the already existent poly-Py tract “Py-Py-Py” and interact now with an activated cryptic donor site (AGGT) in the coding region of exon 34. This generates 4 different NOTCH1 splice variants (NOTCH1 152, 145, 143, 143∗) with new boundary sequences where the PEST domain coding sequence gets spliced out (shown in a green frame, bold type indicates reported variants; regular type represents variants discovered by this study). (B) Maximum entropy model (MaxEntScan) scores of canonical SSs located at the introns adjacent to NOTCH1 3′UTR and the corresponding sequences uploaded for score prediction are shown. (C) Distribution of MaxEntScan scores for NOTCH1 canonical SSs. Sequences used for MaxEntScan prediction are listed in supplemental Table 4. The average scores of canonical NOTCH1 5′SS (blue dots) and 3′SS (red dots) are calculated as 8.23 and 9.20, respectively. (D) Color coding of NOTCH1 3′UTR cryptic acceptor sites generated by NOTCH1 noncoding mutations. Prevalent NOTCH1 152 A>G mutation got a MaxEntScan score for 3′SS at 9.77 which is even higher than the average canonical NOTCH1 3′SS value (9.20; orange). Values located between 9.20 and minimum value (4.60) of canonical NOTCH1 3′SS are shown in yellow (NOTCH1 145 A>G and NOTCH1 143∗ A>C). Of note, the NOTCH1 143∗ A>C score was calculated for NOTCH1 141 acceptor and is shown in white type. The NOTCH1 143 A>G value is higher than the reported cutoff value of SS 3.0,23 and is shown in red type. Py, pyrimidine.
Figure 1.

In silico analysis predicts aberrant splicing events of NOTCH1 pre-mRNA. (A) Schematic representation of C-terminal NOTCH1 pre-mRNA. NOTCH1 wt mRNA with the PEST domain coding sequence in exon 34 is spliced canonically (left), while upon mutation alternative splicing is induced (right). Here, the mutated 3′UTR sequences as indicated create acceptor sites, which can form a stronger acceptor motif with the already existent poly-Py tract “Py-Py-Py” and interact now with an activated cryptic donor site (AGGT) in the coding region of exon 34. This generates 4 different NOTCH1 splice variants (NOTCH1 152, 145, 143, 143∗) with new boundary sequences where the PEST domain coding sequence gets spliced out (shown in a green frame, bold type indicates reported variants; regular type represents variants discovered by this study). (B) Maximum entropy model (MaxEntScan) scores of canonical SSs located at the introns adjacent to NOTCH1 3′UTR and the corresponding sequences uploaded for score prediction are shown. (C) Distribution of MaxEntScan scores for NOTCH1 canonical SSs. Sequences used for MaxEntScan prediction are listed in supplemental Table 4. The average scores of canonical NOTCH1 5′SS (blue dots) and 3′SS (red dots) are calculated as 8.23 and 9.20, respectively. (D) Color coding of NOTCH1 3′UTR cryptic acceptor sites generated by NOTCH1 noncoding mutations. Prevalent NOTCH1 152 A>G mutation got a MaxEntScan score for 3′SS at 9.77 which is even higher than the average canonical NOTCH1 3′SS value (9.20; orange). Values located between 9.20 and minimum value (4.60) of canonical NOTCH1 3′SS are shown in yellow (NOTCH1 145 A>G and NOTCH1 143∗ A>C). Of note, the NOTCH1 143∗ A>C score was calculated for NOTCH1 141 acceptor and is shown in white type. The NOTCH1 143 A>G value is higher than the reported cutoff value of SS 3.0,23 and is shown in red type. Py, pyrimidine.

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