Fig. 4
Analysis of the TSS variants of 53BP1. a Organisation of the LP/SP reporter (see also Additional file 1: Figure S6). The reporter carries two overlapping ORFs, LP (indicated as a yellow rectangle) and SP (indicated as a grey rectangle) each carrying a HA epitope tag (plus an epitope tag unique to each, namely FLAG and MYC). The TL variant was inserted upstream of the AUGGENE. Immunoblotting with the anti-HA Ab permits monitoring of multiple independent initiation events at the AUG codons indicated. b LP/SP reporters carrying the 53BP1 TL variants V1/2 and V3 were transiently expressed in both MCF7 and MCF10A cells (duplicate independent transfections). Proteins were resolved by SDS-PAGE and analysed by immunoblotting using the anti-HA Ab (upper image). The blots were quantitated and the intensity of the protein band corresponding to each initiation site was evaluated. This is plotted in the lower image as the average of the duplicate values (the intensity is an arbitrary value). Each AUG codon in each cellular context is colour coded (e.g., V1/2 in MCF7 cells is green and in MCF10A cells is blue). The pattern of initiation events measured for the two variants in the same cell background, or the same variant in the different cell backgrounds are indicated. c The upper image indicates polysomal profiles for the two cell types with the light and heavy polysomal fractions indicated. RT-PCR was performed across the gradient using primer sets specific for each TL variant plus total 53BP1. The amplicons were resolved on a polyacrylamide gel (middle image) which was then quantitated and plotted graphically (lower image). d Schematic representation indicating the positioning of AUG codons within the 5′ end of the 53BP1 transcript. The blue rectangle represents the 53BP1 ORF. The green rectangle represents an internal overlapping ORF located within the 5′ end of the transcript. The location of AUG initiation codons whose positions relative to the AUG53BP1 roughly correspond to the AUGa/b/c and AUGLP sites in the reporter (Panel a, see also Additional file 1: Figure S6A) are also indicated. The brackets indicate the number of amino acids (aas) deleted from the 53BP1 protein should these initiation codons drive expression. The 5′ TL is indicated as a horizontal red line upstream of the AUG53BP1. The positioning of the small uORF in V3, is indicated as a grey rectangle below the red line since it is not in the same reading frame as 53BP1. The length of the uORF is indicated as number of codons. Note that the 5′ TL of V1/2 possesses no uORFs