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[Preprint]. 2023 Jul 10:2023.07.10.548208. [Version 1] doi: 10.1101/2023.07.10.548208

Extended Data Fig. 3 |. AML1/ETO splice product generated from co-expressed CAGE modules in cells.

Extended Data Fig. 3 |

a, Protein splicing junctions employed in the generation of AML1/ETO using the caged Npu and NrdJ-1 split inteins. The caged Npu system required the insertion of a ‘CF’ di-peptide sequence to facilitate efficient protein trans-splicing. This ‘scar’ is added to the N-terminus of ETO segment. By contrast, the caged NrdJ-1 split intein supports traceless splicing using the native serine at position 36 of ETO. b, Immunoblots of HA-tagged AML1/ETO splice product (SP) generated in HEK293T cells co-expressing AML1-CAGEN (Myc-tag) and CAGEC-ETO (HA-tag). Cells were treated with either DMSO or rapalog (100 nM) for 18 hours before analysis. HEK293T cells expressing a HA-tagged AML1/ETO fusion (WT) are used as a positive control (right most lane). c, Immunoblots of HA-tagged AML1/ETO splice product (SP) generated HEK293T cells co-expressing AML1-CAGEN (wild-type or inactive C1A mutant) and CAGEC-ETO. Cells were treated with either DMSO or rapalog (100 nM) for 18 hours before analysis. d, e, Characterization of the AML1/ETO splice product by mass spectrometry. AML1/ETO splice product generated in HEK293T cells was subjected to trypsinolysis and targeted proteomics used to identify the tryptic peptide spanning the splice junction (Calculated m/z = 519.517, z = 4). Overlaid PRM chromatograms for the six parent-to-daughter transitions in targeted proteomics runs (d) and annotated MS/MS spectrum from the peptide (e). f, Immunoblots of AML1/ETO generation in HEK293T cells co-expressing AML1-CAGEN and CAGEC-ETO. Cells were treated with DMSO or increasing doses of rapalog for 18 hours before analysis. g, h, Immunoblots of AML1/ETO generation in HEK293T cells co-expressing AML1-CAGEN and CAGEC-ETO. Cells were treated with DMSO or rapalog (100 nM) for indicated time-points before analysis. i, Subcellular location of the AML1-CAGEN (C1A mutant) and CAGEC-ETO in HEK293T cells treated with DMSO or rapalog (100 nM) for 18 hours before immunoblotting analysis. The C1A mutation of the CAGEN module blocks the splice product generation. CAGEN refers to ‘NrdJ-1NCage-FKBP-NES’, and CAGEC refers to ‘NES-FRB-NrdJ-1CCage’. Data in (b-c) and (f-i) are representative of n=3 independent experiments. In panels (b-h), CAGEN refers to ‘NpuNCage-FKBP-NES,’ and CAGEC refers to ‘NES-FRB-NpuCCage.