Extended Data Fig. 6: IPA-generated truncated proteins resemble the protein products of truncating DNA mutations and have cancer-promoting properties.
From: Widespread intronic polyadenylation inactivates tumour suppressor genes in leukaemia
a, CARD11 IPA results in translation of intronic nucleotides (grey) until an in-frame stop codon is encountered. This results in the generation of 16 new amino acids (grey) downstream of exon 10. In the case of MGA IPA, three new amino acids downstream of exon 9 are generated. b, Western blot showing that TMD8 cells express similar amounts of CARD11 IPA as CLL samples. The western blot is shown as in Fig. 2a and was performed twice. Actin was used as loading control. c, Western blot (as in b) showing full-length CARD11 as well as CARD11 IPA in TMD8 cells expressing a control shRNA (Co), an shRNA that exclusively knocks down the full-length protein and two different shRNAs that exclusively knock down the CARD11 IPA isoform. The experiment was performed twice with similar results. GAPDH was used as loading control. d, Endogenous phospho-NF-κB p65 levels were measured by FACS in TMD8 cells expressing the indicated shRNAs from c. Mean fluorescent intensity values are shown in parentheses in FACS plots of a representative experiment out of three. e, Immunoprecipitation of V5-DICER or V5-DICER IPA from HEK293T cells using an anti-V5 antibody. The experiment was performed twice with similar results. 2.5% of input was loaded. f, The extent of miRNA processing depends on the expression levels of full-length DICER, but not IPA. Shown are wild-type (WT) and DICER knockout (KO) HCT116 cells. Re-expression of different amounts of full-length DICER1 protein in the knockout cells (measured by western blot of DICER1 in the top panel) results in different levels of endogenous let-7 expression (measured by northern blot in the bottom panel; compare lanes 3 and 4). Expression of DICER IPA has no influence on miRNA processing (compare lanes 4 and 5). Actin and U6 were used as loading controls. The experiment was performed twice with similar results. g, Western blot of MGA. MGA and MGA IPA were cloned and expressed in HEK293T cells to confirm the predicted protein size. The experiment was performed twice with similar results. Shown is also the endogenous MGA expression in Raji cells. Actin was used as loading control on the same blot. Asterisk denotes an unspecific band. h, Protein models of full-length and FOXN3 IPA are shown as in Fig. 2b. The IPA-generated protein truncates the fork-head domain and is predicted to lose the repressive activity. i, As in a, but for FOXN3. FOXN3 IPA generates 32 new amino acids downstream of exon 2. j, FOXN3 IPA significantly derepresses expression of the oncogenic targets MYC and PIM2. Fold change in mRNA level of endogenous genes in MEC1 B cells after transfection of GFP–FOXN3 IPA compared with transfection of full-length GFP-FOXN3. HPRT-normalized values are shown as box plots (as in Fig. 1e) from n = 5 biologically independent experiments, each performed in technical triplicates. **P = 0.002, two-sided t-test for independent samples. For gel source data, see Supplementary Fig. 1.