PARP1 negatively regulates MAPK signaling by impairing BRAF-X1 translation
Journal of Hematology & Oncology volume 16, Article number: 33 (2023)
In human cells BRAF oncogene is invariably expressed as a mix of two coding transcripts: BRAF-ref and BRAF-X1. These two mRNA isoforms, remarkably different in the sequence and length of their 3′UTRs, are potentially involved in distinct post-transcriptional regulatory circuits. Herein, we identify PARP1 among the mRNA Binding Proteins that specifically target the X1 3′UTR in melanoma cells. Mechanistically, PARP1 Zinc Finger domain down-regulates BRAF expression at the translational level. As a consequence, it exerts a negative impact on MAPK pathway, and sensitizes melanoma cells to BRAF and MEK inhibitors, both in vitro and in vivo. In summary, our study unveils PARP1 as a negative regulator of the highly oncogenic MAPK pathway in melanoma, through the modulation of BRAF-X1 expression.
To the editor
Although BRAFV600E oncogenic kinase is extensively studied as cancer driver and represents a valuable therapeutic target, the regulation of BRAF gene expression remains largely unknown . Recently, we reported that, irrespectively of its mutational status, BRAF is expressed as a mix of two different splicing variants, namely BRAF-ref and BRAF-X1. These mRNA isoforms are characterized by 3′UTRs of different sequence and length (~ 100nt vs. ~ 1300 to 7000nt) . The corresponding protein isoforms differ at the C-terminal, however they are both endowed with kinase activity and together account for BRAFV600E oncogenic features in melanoma cells [2, 3].
A very long 3′UTR such as the X1 calls for post-transcriptional regulation. Indeed, we have already identified quite a large group of X1-targeting microRNAs that positively or negatively affect RNA stability and translation . Since increasing evidence links RBPs with tumorigenesis , we performed a high-throughput screening for X1-targeting mRBPs. Such screening led us to the identification of PARP1 as negative regulator of BRAF-X1 translation and, consequently, of MAPK pathway signaling.
We performed REMSA on the ~ 1300nt long version of X1 3′UTR, using S100 cytoplasmic protein extract obtained from A375 melanoma cell line. We observed an X1-specific band shift when a radiolabeled riboprobe corresponding to the last 186nt of the 3′UTR was used (R8 probe in Fig. 1a; see also Additional file 1, Additional file 2: Figs. S1–S3 and Additional file 3: Table S1). Subsequently, we performed a pull-down experiment incubating A375 S100 extract with a desthiobiotinylated R8 riboprobe, and 87 cytoplasmic proteins that specifically bind to the probe were identified by mass spectrometry. Among these proteins, we selected 51 for which no peptides were found in control samples (pull-down performed with a riboprobe of unrelated sequence; see Fig. 1b and Additional file 4: Table S2). STRING analysis revealed that these 51 proteins form a highly interconnected network (Fig. 1c) and belong to pathways related to RNA metabolism (Additional file 5: Table S3). The subset of 20 proteins classified as mRBPs (, red nodes in Fig. 1c; see also Additional file 2: Fig. S4) were further characterized using GEPIA database (http://gepia.cancer-pku.cn/detail.php?gene =). 6 mRBPs out of 20 have a prognostic value in melanoma (Additional file 2: Fig. S5), and 5 show a positive correlation with BRAF mRNA levels (DHX36, ILF3, KHSRP, PARP1 and STRAP, Additional file 2: Fig. S6). According to two databases (ARED (https://brp.kfshrc.edu.sa/ARED/) and AREsite2 (http://rna.tbi.univie.ac.at/AREsite2/welcome)), the R8 fragment of X1 3′UTR does not contain AU-Rich Elements. Therefore, known AUBPs such as ILF3 and KHSRP were not prioritized for further analysis. The binding affinity of the remaining 3 proteins (DHX36, PARP1, and STRAP) with R8 fragment of X1 3′UTR was predicted and ranked using the catRAPID omics v2.0 program. As shown in Fig. 1d and Additional file 6: Table S4, the affinity of PARP1 is top-scoring.
PARP1 has been intensively studied and therapeutically exploited as a nuclear enzyme involved in recognition and repair of DNA damage. However, it has recently emerged as a multifaceted post-transcriptional regulator , also considering its partially cytoplasmic localization (Additional file 2: Figs. S7, S8) and its ability to bind mature poly(A) + mRNA . After demonstrating that the binding of PARP1 protein to X1 3′UTR is direct (Fig. 1e), we explored the consequences of such binding, in terms of BRAF mRNA/protein levels and MAPK signaling.
Using appropriate Luciferase reporters (Fig. 1f, left and middle) and western blot analysis of A375 (BRAFV600E homozygous) and 501Mel (BRAFV600E heterozygous) melanoma cell lines (Fig. 1g, left), we observed that PARP1 knock down by siRNA leads to an increase in Luciferase activity of full length X1 reporter, and in endogenous BRAF protein levels, respectively. Conversely, PARP1 stable overexpression by means of inducible pCW-PARP1 vector (Additional file 2: Fig. S9, see also ) leads to opposite results (Fig. 1f, right and Fig. 1g, right). Interestingly, in Additional file 2: Fig. S10 we show that the negative regulation exerted by PARP1 on BRAF persists in a context of acquired resistance to vem. Next, we investigated whether PARP1 affects X1 mRNA or protein. We found that PARP1 does not alter X1 mRNA levels nor stability (Additional file 2: Figs. S11, S12), and rather impairs X1 mRNA translation (Fig. 1h). Consistently, the K222I mutant, which causes nuclear exclusion (Additional file 2: Fig. S13 and S14a,b), allowed us to confirm that it is cytoplasmic PARP1 to act as negative regulator of BRAF (Additional file 2: Fig. S14c).
To validate our findings using melanoma patient data, we resorted to the TCGA-SKCM dataset. Specifically, we compared BRAF mRNA and protein level in the 25% high PARP1 protein expressors versus the 25% low PARP1 protein expressors. Notably, no differences in BRAF mRNA level are present between the two groups (Fig. 1i), but the 25% high PARP1 protein expressors show lower BRAF protein levels, in accordance with our in vitro data (Fig. 1j, see also Additional file 2: Fig. S15). All together these data indicate that cytoplasmic PARP1 directly binds to the R8 region and represses BRAF-X1 translation.
PARP1 consists of three functional domains: the DNA/RNA Binding domain, which in turn is composed of 3 Zinc Finger motifs (Zn), the Auto-modification domain (Auto) and the Catalytic (parylating) domain (Cat) (Fig. 2a). To pinpoint which domain is responsible for BRAF-X1 regulation, we stably overexpressed each of them separately, by means of inducible pCW-HA vectors (Fig. 2a and Additional file 2: Fig. S13). Interestingly, we found that the Zn domain, which maintains similar intracellular localization as full length PARP1 (Additional file 2: Fig. S16), recapitulates the effect of full length protein in terms of decrease in Luciferase activity (Fig. 2b), decrease in endogenous BRAF protein level (Fig. 2c), and translation impairment (Fig. 2d). We modeled the complex that PARP1 protein forms with R8 RNA fragment and found two interaction sites that fall within the Zn domain (Fig. 2e and Additional file 2: Fig. S17). In addition, the interaction between Zn domain and X1 3′UTR was confirmed experimentally, by performing RIP-qRT-PCR analysis on the Zn domain (Fig. 2f). Interestingly, a PARP1 catalytic inhibitor such as Olaparib does not affect endogenous BRAF protein level (Additional file 2: Figs. S18, S19). All together, these results indicate that PARP1 requires the Zn domain to bind X1 mRNA and that, contrary to what is known so far about PARP1-mediated regulation of mRNA stability , parylation is completely dispensable for PARP1-mediated regulation of X1 mRNA translation.
BRAFV600E-X1 downregulation results in a reduction in pMEK, which ultimately affects cellular features such as proliferation and migration . Consistently, we observed that the over-expression of PARP1 Zn domain decreases pMEK levels (Fig. 2g), and impairs cell proliferation (Fig. 2h), as well as cell motility in vitro (Fig. 2i and Additional file 2: Fig. S20) and in a xenograft model in zebrafish (Fig. 2j). Crucially, by detecting no change in γ-H2AX foci, we excluded that Zn domain acts as a dominant negative mutant that is trapped on DNA and triggers DNA damage  (Fig. 2k and Additional file 2: Fig. S21). Conversely, in agreement with its role as negative regulator of the MAPK pathway, we found that the Zn domain increases vem-induced ROS (Fig. 2l), hence sensitizes melanoma cells to vem, cob and vem + cob treatment, both in vitro (Fig. 2m, n) and in vivo in a zebrafish xenograft model (Fig. 2o).
In summary, we show that cytoplasmic PARP1 binds BRAF-X1 mRNA and negatively regulates translation (Fig. 2p), in a 3′UTR binding-dependent, but parylation-independent manner. On one side, our results further consolidate the notion that the X1 variant of BRAF is subjected to a tight post-transcriptional regulation, which ultimately has an impact on the output of MAPK pathway. On the other side, our results provide the first example of a specific mRNA whose translation is directly affected by PARP1 mRNA binding activity . Considering that such an mRNA is BRAF-X1, they also unveil how PARP1 sphere of influence extends to the regulation of MAPK pathway, hence prompt to explore whether PARP1 is involved in the pathogenesis of those tumor types that are driven by the hyperactivation of such pathway . In more general terms, our data uncover the oncosuppressive liaison existing between BRAF-X1 3′UTR and PARP1 Zn domain, challenging the definition of BRAFV600E and PARP1 as pure oncogenes that cooperate in melanocyte transformation .
Availability of data and materials
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
3′ UnTranslated Region
AU-Rich Binding Protein
Hours post fertilization
Inhibitors of the MAPK pathway
mRNA Binding Protein
RNA Electrophoretic Mobility Shift Assay
Reactive Oxygen Species
Relative Translational Efficiency
- SKCM dataset:
SKin Cutaneous Melanoma dataset
The Cancer Genome Atlas
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The authors thank A. Mercatanti for siPARP1 design and all Poliseno lab members for helpful discussions.
This work was supported by ISPRO-Istituto per lo Studio, la Prevenzione e la Rete Oncologica [institutional funding to LP] and SATIN-POR CAMPANIA FESR 2014/2020 to GDP. It was also partially supported by AIRC-Associazione Italiana Ricerca sul Cancro [MFAG #17095 and IG #25694 to LP]. During the revision of the article, Andrea Marranci was supported by Fondazione Umberto Veronesi.
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Supplementary Material: REMSA analysis.
: The 87 cytoplasmic proteins identified by mass spectrometry.
: Reactome pathways to which the 51 selected RBPs belong, listed top to bottom according to increasing FDR values.
: Binding affinity of the 20 mRBPs to the R8 fragment of X1 3’UTR, according to catRAPID omics v2.0 program.
Material and Methods.
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Marranci, A., Prantera, A., Masotti, S. et al. PARP1 negatively regulates MAPK signaling by impairing BRAF-X1 translation. J Hematol Oncol 16, 33 (2023). https://doi.org/10.1186/s13045-023-01428-2
- MAPK pathway