Victoria Beringer

RNA-binding proteins (RBPs) are essential regulators of RNA metabolism and gene expression, influencing processes such as splicing, stability, localization, and translation. Despite their critical roles in health and disease, including cancer, identifying RNA–protein interactions remains challenging due to technical limitations and biases of existing methods. Here we review and compare experimental techniques—including in vitro affinity purification, in vivo crosslinking, and proximity labeling—and computational prediction tools for RBP identification. We assess their strengths, limitations, and applicability across biological contexts, emphasizing the benefits of integrating experimental and computational strategies. Our analysis provides practical guidelines for selecting appropriate methodologies tailored to different cell types and research goals. These insights aim to facilitate more accurate mapping of RNA–protein interactomes, thereby advancing understanding of RBP functions and supporting the development of novel therapeutic interventions targeting RNA–protein complexes.

RNA-binding proteins (RBPs) are essential regulators of RNA metabolism and gene expression, influencing processes such as splicing, stability, localization, and translation. Despite their critical roles in health and disease, including cancer, identifying RNA-protein interactions remains challenging due to technical limitations and biases of existing methods. Here we review and compare experimental techniques—including in vitro affinity purification, in vivo cross-linking, and proximity labeling—and computational prediction tools for RBP identification. We assess their strengths, limitations, and applicability across biological contexts, emphasizing the benefits of integrating experimental and computational strategies. Our analysis provides practical guidelines for selecting appropriate methodologies tailored to different cell types and research goals. These insights aim to facilitate more accurate mapping of RNA-protein interactomes, thereby advancing understanding of RBP functions and supporting the development of novel therapeutic interventions targeting RNA-protein complexes.

Background: Ewing’s sarcoma (EwS) is a pediatric bone and soft tissue cancer driven by the oncogenic fusion protein EWS::FLI1. Currently, EwS lacks targeted therapies, necessitating the identification of novel regulatory mechanisms. While the role of microRNAs and long non-coding RNAs has been explored in EwS, the presence and functional significance of circular RNAs (circRNAs) in EwS is not reported. This is the first study to report the presence and role of oncogenic circRNA, circZNF609 in EwS tumor progression. Methods: Expression of circZNF609 was validated in 5 different EwS cell lines using qPCR. Cellular localization of circZNF609 was identified using circFISH. Functional assays for proliferation, migration and apoptosis were performed in wild type and circZNF609 knocked down (KD) cell lines to confirm its oncogenic role. The impact of circZNF609 on EWS::FLI1 protein levels was confirmed using western blots, immunofluorescence, and polysome fractionation. Mechanistic insights were gained utilizing bioinformatic, dual-luciferase reporter assays, rescue experiments, and microscopy to identify and validate the circRNA-miRNA-mRNA regulatory axis. Results: We report the first identification of circZNF609 in EwS, demonstrating that its expression is EWS::FLI1-dependent. Functional analysis reveals that circZNF609 promotes cell proliferation and metastasis while inhibiting apoptosis. Mechanistically, circZNF609 acts as a molecular sponge for miR-145-5p. By sequestering this miRNA, circZNF609 prevents the translational repression of EWS::FLI1, thereby sustaining oncogenic signaling. Conclusions: These findings identify circZNF609 as a novel post-transcriptional regulator of EWS::FLI1 and establish its critical role in EwS pathogenesis. Our results suggest that targeting the circZNF609/miR-145-5p/EWS::FLI1 axis may offer a promising therapeutic strategy for EwS.

Ewing’s sarcoma (EwS) is a pediatric bone and soft tissue cancer driven by the oncogenic fusion protein EWS::FLI1, with no current targeted therapies. Although the role of some of the non-coding RNAs (ncRNAs) including microRNAs and long non coding RNAs have been explored in EwS, no work is reported on presence and role of newly appreciated class of regulatory RNAs called circular RNAs (circRNA). Our work here reports the first identification and functional characterization of the role of circRNA circZNF609 in EwS. Here we identify and characterize circZNF609, a circRNA expressed in an EWS::FLI1-dependent manner, which promotes EwS cell proliferation, metastasis, and inhibits apoptosis. Mechanistically, circZNF609 acts as a sponge for miR-145-5p, preventing miRNA-mediated translational repression of EWS::FLI1, thereby sustaining oncogenic signaling. Inhibition of miR-145-5p partially rescues the effects of circZNF609 knockdown, confirming this regulatory axis. These findings reveal circZNF609 as a novel post-transcriptional regulator of EWS::FLI1 and suggest its potential as a therapeutic target in EwS.