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Melanoma RBPome identification reveals PDIA6 as an unconventional RNA-binding protein involved in metastasis

ABSTRACT: RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of two melanoma cell lines with different aggressiveness revealed prevalent changes in RNA binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression and map its RNA-binding domain, which we find to be required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells

ORGANISM(S): Homo sapiens

PROVIDER: GSE174821 | GEO | 2022/07/11

REPOSITORIES: GEO

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Project description:Here, we performed RNA-interactome capture (RIC) on nuclear fractions from human embryonic stem cells (hESCs). The poly(A)+ RNA-bound proteome was determined by UV light-mediated cross-linking (CL) of RNAs to proteins in living cells, followed by nuclei isolation, oligo(dT) purification of poly(A)-RNA-protein complexes, and mass spectrometry analysis of captured proteins. As a control, we applied a similar strategy to non-cross-linked (non-CL) samples. RIC was performed in four independent biological replicates. This data accompanies the manuscript: "Uncovering the RNA-binding protein landscape in the pluripotency network of human embryonic stem cells". Abstract: "Embryonic stem cell (ESC) self-renewal and cell-fate decisions are driven by a broad array of molecular signals. While transcriptional regulators have been extensively studied in human ESCs (hESCs), the extent to which RNA-binding proteins (RBPs) contribute to human pluripotency remains unclear. Here, we carry out a proteome-wide screen and identify 810 proteins that directly bind RNA in hESCs. We reveal that RBPs are preferentially expressed in hESCs and dynamically regulated during exit from pluripotency and early lineage specification. Moreover, we show that nearly 200 RBPs are affected by knockdown of OCT4, a master regulator of pluripotency, several dozen of which are directly bound by this factor. Intriguingly, over 20 percent of the proteins detected in our study are putative DNA- and RNA-binding proteins (DRBPs), among them key transcription factors (TFs). Using fluorescently labeled RNA and seCLIP (single-end enhanced crosslinking and immunoprecipitation) experiments, we discover that the pluripotency-associated STAT3 and OCT4 TFs interact with RNA in hESCs and confirm the direct binding of STAT3 to the conserved NORAD long-noncoding RNA. Taken together, our findings indicate that RBPs have a more widespread role in human pluripotency than previously appreciated, reinforcing the importance of post-transcriptional regulation in stem cell biology".

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Melanoma RBPome identification reveals PDIA6 as an unconventional RNA-binding protein involved in metastasis

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