lncRNA RPPH1 in melanoma: novel functions as cargo of tumor-derived Extracellular Vesicles

The long non-coding RNA (lncRNA) RPPH1 is a ribozyme, an RNA molecule with enzymatic activity, and serves as the catalytic component of the RNase P ribonucleoprotein complex. This complex performs the endonucleolytic cleavage of precursor tRNAs at their 5′ leader sequences. Increasing evidence suggests that RPPH1 may also have non-canonical roles in tumor progression, including its presence as an extracellular vesicles (EVs)-associated RNA species. In a recent study, we reported a significant enrichment of lncRNA RPPH1 in melanoma-derived EVs, both in zebrafish and human models. Tumor progression depends on dynamic interactions between cancer cells and their surrounding tumor microenvironment (TME). Among the mediators of this communication, EVs have emerged as key carriers of bioactive molecules capable of modulating multiple aspects of cancer biology: tumor-derived EVs can influence tumor initiation, growth, and metastatic dissemination. Recent evidence indicates that the RNA cargo contained within these vesicles contributes to pro-tumorigenic inflammation by activating interferon-mediated immune responses through Pattern Recognition Receptor (PRR) signalling pathways. Notably, the injection of melanoma-derived EVs or synthetic RPPH1 into healthy zebrafish larvae induces sterile inflammation, consistent with the activation of PRR-dependent signalling in macrophages. In this PhD work, we investigated the mechanisms underlying the packaging of RPPH1 into extracellular vesicles in melanoma. We demonstrated that RPPH1 is upregulated in melanoma cells and abnormally localizes to the cytoplasm. Proteomic analysis of RPPH1-associated protein partners in melanoma revealed potential novel functions for this lncRNA beyond its canonical role in the RNase P complex, particularly in RNA metabolism. These findings suggest that such alternative functions may contribute to its selective encapsulation within EVs. Using both human and zebrafish melanoma models, we further explored the mechanisms through which melanoma-derived EVs induce sterile inflammation. Our results showed that these EVs, likely through their RPPH1 cargo, activate the RIG-I-like receptor (RLR) pathway in recipient macrophages, triggering an inflammatory response characterized by the upregulation of interferon-stimulated genes (ISGs) and pro-inflammatory cytokines. Single-cell RNA sequencing revealed that this EV-mediated signalling drives the transcriptional reprogramming of macrophages toward a tumor-supportive, pro-inflammatory phenotype, typical of tumor-associated macrophages (TAMs). Notably, this response appears to be primarily confined to a macrophage subpopulation inherently predisposed to endocytosis and EV-trafficking. Overall, these findings contribute to a deeper understanding of how melanoma cells promote tumor progression, reprogramming the immune cells in the tumor microenvironment through EV-mediated RNA signalling.