Fig. 2

PARP1 Zn domain is responsible for the negative regulation of BRAF-X1 translation, and inhibits MAPK pathway in vitro and in vivo. a Schematic representation of the functional domains of PARP1 protein (3042 bp, 1014 aa): DNA/RNA Binding domain, which in turn is composed of 3 Zinc Finger motifs (Zn, green); Auto-modification domain (Auto, gray); Catalytic (parylating) domain (Cat, blue). b Luciferase assay in A375 cells. Among the functional domains tested (pCW-HA-Zn (green), pCW-HA-Auto (gray) and pCW-HA-Cat (blue)), only the overexpression of the Zn domain recapitulates the decrease in Luciferase activity of pMIR-X1-3′UTR plasmid, as observed with full length pCW-PARP1 (orange). The assay was performed 48 h after the transfection of the Luciferase plasmids in cells stably infected with the indicated pCW(-HA) vectors, and the induction of protein overexpression with 2ug/ml doxycycline. c Western blot analysis of BRAF protein level in A375 cells stably infected with pCW-CTRL, pCW-HA-Zn, pCW-HA-Auto, pCW-HA-Cat and pCW-PARP1 vectors, 48 h after induction with 2ug/ml doxycycline. A representative western blot result (left) and bands quantification (right) are shown. d The RTE of pMIR-X1-3′UTR plasmid was calculated in A375 cells stably infected with pCW-CTRL or pCW-HA-Zn, 48 h after transfection of Luciferase plasmids and induction with 2ug/ml doxycycline. e Structural model of PARP1 domain in complex with R8 RNA fragment. PARP1 is represented as surface, while R8 is represented as cartoon. The Zn domain (residues 1–353) is green, the Auto-modification domain (residues 389–643) is gray and the Catalytic domain (residues 662–1014) is blue. The other residues that do not belong to one of these three domains are purple. R8 phosphate-deoxyribose backbone is cyan, while the color code for nitrogenous bases is as follows: A red; G light green; C yellow; U light blue. f RIP-qRT-PCR assay. A375 cells, stably infected with pCW-CTRL or pCW-HA-Zn, were subjected to RIP-qRT-PCR 48 h after induction with 2ug/ml doxycycline. RIP was performed with anti-HA-tag sepharose beads and was coupled with qRT-PCR quantification of BRAF-ref and BRAF-X1 mRNA. g Western blot analysis of BRAF and its downstream effector pMEK in A375 cells stably infected with pCW-CTRL or pCW-HA-Zn, 48 h after induction with 2ug/ml doxycycline. A representative western blot result (left) and bands quantification (right) are shown. h Proliferation assay of A375 and 501Mel cells stably infected with pCW-CTRL or pCW-HA-Zn, 7 days after induction with 2ug/ml doxycycline. i Wound closure assay of A375 cells stably infected with pCW-CTRL or pCW-HA-Zn, 48 h after induction with 2ug/ml doxycycline. j Representative pictures (top), size (bottom left) and distance from injection site (bottom right) of metastases developed in a xenograft model in zebrafish embryos. A375 cells, stably infected with pCW-CTRL or pCW-HA-Zn, were resuspended in PBS and were injected in 48hpf embryos. Then, embryos were allowed to grow for 96 h in E3 medium supplemented with 2ug/ml doxycycline. At the end of this period, the size of red cell masses and their distance from injection site were measured. Scale bar: 300um. k Percentage of γ-H2AX positive cells. γ-H2AX foci, which mark DNA damage, were stained in A375 cells stably infected with pCW-CTRL or pCW-HA-Zn, 48 h after induction with 2ug/ml doxycycline. pCW-CTRL infected cells were concomitantly treated with the indicated concentrations of Olaparib. See Additional file 2: Fig. S21 for representative pictures of each experimental condition. l Total ROS levels measured in A375 cells stably infected with pCW-CTRL, pCW-HA-Zn or pCW-PARP1, after 48 h of induction with 2ug/ml doxycycline and concomitant treatment with 2uM vem. m Growth curve of A375 cells stably infected with pCW-CTRL, pCW-HA-Zn or pCW-PARP1, after 7 days of induction with 2ug/ml doxycycline and concomitant treatment with the indicated concentrations of vem. n Proliferation assay of A375 cells stably infected with pCW-CTRL, pCW-HA-Zn or pCW-PARP1, 14 days after induction with 2ug/ml doxycycline and treatment with the indicated concentrations of vem, cob or vem + cob. o Area of tumors developed in a xenograft model in zebrafish embryos. A375 cells, stably infected with pCW-CTRL or pCW-HA-Zn, were injected in 48hpf embryos. Then, embryos were allowed to grow for 48 h in E3 medium supplemented with 2ug/ml doxycycline and 2uM vem. At the end of this period, the area of red cell masses was quantified. Representative pictures (left) and the results of area quantification (right) are shown. Scale bar: 300um. Graphs represent the mean ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. p Cartoon summarizing our findings. PARP1 is a new player in the regulation of the highly oncogenic MAPK pathway in melanoma. Through the mRNA binding activity of its Zn domain, it negatively regulates the translation of BRAF-X1 isoform, leading to a decrease in MAPK signaling and, consequently, a decrease in cell proliferation/motility accompanied by an increase in sensitivity to MAPKi. Cartoon created with BioRender.com