Fig. 3

A transposon-based biosensor screen to identify PARylated proteins. a A genetic screen to identify PARylation events. (i) UPATrap-VC was introduced into CAL51 cells, generating six tagged cell libraries (5000 clones each). (ii) Either PBZ-GF, PBZ-VN, or PBZ-4A-VN biosensors were introduced into each library and GFP-positive cells were isolated (iii); PBZ-GFP showed 60% GFP-positive cells, while the PBZ-VN constructs showed 0.1% GFP-positive cells. (iv) Genomic DNA was isolated from GFP-positive cells and UPATrap-VC integration sites identified by non-restrictive linear amplification PCR (nrLAM-PCR) followed by deep-sequencing. Each gDNA sample was amplified and sequenced in three independent reactions (A, B, C). b A schematic of the transposon-mediated VC tagging, when an UPATrap-VC transposon is introduced into genes. Yellow triangles Tol2 transposon repeats, SA splicing acceptor, SD splicing donor, IRES internal ribosome entry site, NeoR G418-resistance gene, pA polyAdenylation signal. Integration of UPATrap-VC into genes results in the production of protein-VC fusion proteins and NeoR protein. c Integration of a full-length GFP version of UPATrap (UPATrap-GFP) generates specific localization pattern in different GFP-positive colonies. This suggests that the transposon has captured and generated in-frame protein-VC fusion in a specific gene. d nrLAM-PCR and deep-sequencing from independent reactions is highly reproducible. Scatter plots are shown illustrating the correlation between unique read depth from three replica nrLAM-PCR and sequencing reactions (A, B, and C); Spearman’s rank correlation > 0.98. e Distribution of sequencing depth across all libraries in the screen. Approximately 50 genomic sites were represented by a unique read depth of > 30 reads (see detailed description in methods). f A schematic representation of the three genes identified with two, independent, transposon integration sites (indicated by red circles). g Biosensor signal obtained by the expression of full-length NPM1-VC and CTIF-VC in combination with PNZ-VN. The cells were co-stained with antibodies, recognizing the endogenous NPM1 and CTIF, respectively. h PARylation biosensor screen detects “hits” with different subcellular localization. Confocal imaging of VC-VN GFP signal for 11 genes identified in the screen are shown. NPM1 is a known PARylation target. Scale bars represent 5 µm