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

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Extended Data Fig. 4 | — a, Protein splicing junctions employed in the generation of MLL/AF9 using the caged Npu and NrdJ-1 split inteins. The caged Npu system required the insertion of a ‘CFN’ tri-peptide sequence to facilitate efficient protein trans-splicing. This ‘scar’ is added to the N-terminus of AF9 segment. The caged NrdJ-1 split intein supports traceless splicing using the native serine at position 490 of AF9. b, Immunoblots of MLL/AF9 (HA-tag) splice product (SP) generation in HEK293T cells co-expressing MLL-CAGE^N (Myc-tag) and CAGE^C-AF9 (HA-tag). Cells were treated with DMSO or rapalog (100 nM) for indicated time-points before analysis. CAGE^N refers to ‘NpuN^Cage-FKBP-NES,’ and CAGE^C refers to ‘NES-FRB-NpuC^Cage.’ c, d, Immunoblots of MLL/AF9 generation in HEK293T cells co-expressing MLL-CAGE^N and CAGE^C-AF9. Cells were treated with DMSO or increasing doses of rapalog for 18 hours before analysis. CAGE constructs incorporated either Npu^Cage (c) or NrdJ-1^Cage (d). e, f, Immunoblots of the HA-tagged MLL/AF9 splice product generated in HEK293T cells co-expressing MLL-CAGE^N and CAGE^C-AF9 treated with rapalog (100 nM) for 18 hours followed by a subcellular fraction to separate cytosolic and nuclear proteins. Tubulin and histone H4 serve as cytosolic and nuclear markers, respectively. CAGE constructs incorporated either Npu^Cage (e) or NrdJ-1^Cage (f). g, Protein splicing junctions employed in the generation of NUP98/HOXA9 using the caged Npu and NrdJ-1 split inteins. The caged Npu system required the insertion of a ‘CFN’ tri-peptide sequence to facilitate efficient protein trans-splicing. This ‘scar’ is added to the N-terminus of HOXA9 segment. The caged NrdJ-1 split intein supports traceless splicing using the native serine at position 176 of HOXA9. h, Immunoblots of NUP98/HOXA9 (HA-tag) splice product (SP) generation in HEK293T cells co-expressing NUP98-CAGE^N (Myc-tag) and CAGE^C-HOXA9 (HA-tag). Cells were treated with DMSO or rapalog (100 nM) for indicated time-points before analysis. CAGE^N refers to ‘NpuN^Cage-FKBP-NES,’ and CAGE^C refers to ‘NES-FRB-NpuC^Cage.’ i, j, Immunoblots of NUP98/HOXA9 generation in HEK293T cells co-expressing NUP98-CAGE^N and CAGE^C-HOXA9. Cells were treated with DMSO or increasing doses of rapalog for 18 hours before analysis. CAGE constructs incorporated either Npu^Cage (i) or NrdJ-1^Cage (j). k, l, Immunoblots of the HA-tagged NUP98/HOXA9 splice product generated in HEK293T cells co-expressing NUP98-CAGE^N and CAGE^C-HOXA9 treated with rapalog (100 nM) for 18 hours followed by a subcellular fraction to separate cytosolic and nuclear proteins. Tubulin and histone H4 serve as cytosolic and nuclear markers, respectively. CAGE constructs incorporated either Npu^Cage (k) or NrdJ-1^Cage (l). Data in (b-f) and (h-l) are representative of n=3 independent experiments.