Fig. 1

PARP1 directly binds the 3′UTR of BRAF-X1 mRNA and negatively regulates its translation in melanoma cells. a REMSA. On the top, schematic representation of the radiolabeled riboprobes used for the experiment. On the bottom, band shifts obtained incubating S100 cytoplasmic protein extract obtained from A375 cells with probe R7 and R8 (blue asterisks). See Additional file 2: Fig. S3 for further details. b Summary of the analytic workflow to which we subjected the 87 cytoplasmic proteins obtained by mass spectrometry analysis of R8 pull-down. c Interaction network of the 51 proteins showing no peptides in pull-down control samples, as obtained by STRING analysis. The 51 proteins are represented as nodes of the network, with the 20 mRBPs highlighted in red. The connections among nodes are depicted as lines and their strength as colors: light gray/gray/black corresponds to 0.5 (weak)/0.75 (intermediate)/1 (strong) overall score. Nodes have been arranged according to the number of connections they have with the other nodes of the network. The lower is the number of connections, the more peripheral is the position of a node. The Protein–Protein Interaction (PPI) enrichment p-value of the network is < 1.0E − 16. d Binding affinity of the 20 mRBPs to the R8 fragment of X1 3′UTR, according to catRAPID omics v2.0 program. RNA Fragment of R8: part of the R8 fragment bound by the mRBP. Interaction Propensity: probability of interaction between one protein (or region) and one RNA (or region). Z_score: correction of potential biases originating from the length of the RNAs and impacting the Interaction Propensity. RBP Propensity: measure of the propensity of the protein to bind the RNA (1 if the protein is in the RBP precompiled library). RNA Binding Domains: number of RNA binding domain occurrences found in the protein sequence. RNA Binding Motifs: number of RNA binding motif instances found on the RNA sequence. Conserved Interactions: number of organisms in which the interaction is conserved out of those in which an orthologous pair is found. Ranking: the ranking results from three individual values: (1) catRAPID corrected propensity, (2) RBP propensity, and (3) presence of known RNA Binding Motifs. The 5 top-scoring protein are reported. PARP1 has the highest rank, DHX36 is fourth, while STRAP is 17th (complete list in Additional file 6: Table S4). e Pull-down assay reveals the direct binding between desthiobiotinylated R8 riboprobe and recombinant PARP1 protein. Both the unrelated 3′UTR of Androgen Receptor mRNA, provided by the pull-down kit, and R2 riboprobe, which does not show any band shift when incubated with S100 cytoplasmic protein extract of A375 cells (see also Additional file 1), were used as negative controls. f Luciferase assays in A375 cells. The full-length ref or X1 3ʹUTR, as well as the X1 3′UTR missing the R8 region (X1 ΔR8, left) were cloned downstream of Luciferase CDS in pMIR plasmid, so that pMIR-ref-3′UTR, pMIR-X1-3′UTR and pMIR-X1-3′UTRΔR8 Luciferase plasmids were obtained. Such plasmids were either cotransfected with siCT/siPARP1 in A375 cells (middle), or transfected in A375 cells previously infected with pCW-CTRL/pCW-PARP1 vectors (right). 48 h after transfection, PARP1 knock-down is associated with an increase in the Luciferase activity of pMIR-X1-3′UTR plasmid, but not of pMIR-ref-3′UTR and pMIR-X1-3′UTRΔR8 plasmids, indicating that PARP1 interacts specifically with the X1 3′UTR, and more precisely with the R8 region. Consistently, 48 h after transfection and concomitant induction with 2ug/ml of doxycycline, PARP1 overexpression results in a decrease in the Luciferase activity of pMIR-X1-3′UTR plasmid. g Western blot analysis of BRAF protein level in A375 and 501Mel cells. Western blot was performed 48 h after the transfection of siPARP1 (left), or 48 h after induction of PARP1 overexpression in cells stably infected with pCW-PARP1 vector and treated with 2ug/ml doxycycline (right). A representative western blot result (top) and bands quantification (bottom) are shown. h The RTE of pMIR-X1-3′UTR plasmid, which is the ratio between Luciferase protein activity and Luciferase mRNA level, was calculated in A375 cells, 48 h after the cotransfection of the Luciferase plasmids with siPARP1. Graphs represent the mean ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. i BRAF mRNA expression (RSEM) in the group of high PARP1 protein expressors (25% percentile) versus low PARP1 protein expressors (75% percentile) within the TCGA-SKCM melanoma patient dataset. j BRAF protein expression (RPPA) in the group of high PARP1 protein expressors (25% percentile) versus low PARP1 protein expressors (75% percentile) within the TCGA-SKCM melanoma patient dataset