CircRNAs are a category of regulatory RNAs that have garnered significant attention in the field of regulatory RNA research due to their structural stability and tissue-specific expression. Their circular configuration, formed via back-splicing, results in a covalently closed structure that exhibits greater resistance to exonucleases compared to linear RNAs. The distinctive regulation of circRNAs is closely associated with several physiological processes, as well as the advancement of pathophysiological processes in several human diseases. Despite a good understanding of the biogenesis of circular RNA, details of their biological roles are still being explored. With the steady rise in the number of investigations being carried out regarding the involvement of circRNAs in various regulatory pathways, understanding the biological and clinical relevance of circRNA-mediated regulation has become challenging. Given the vast landscape of circRNA research in the development of the heart and vasculature, we evaluated cardiovascular system research as a model to critically review the state-of-the-art understanding of the biologically relevant functions of circRNAs. We conclude the review with a discussion of the limitations of current functional studies and provide potential solutions by which these limitations can be addressed to identify and validate the meaningful and impactful functions of circRNAs in different physiological processes and diseases.

Cerebral palsy is a set of common, severe, motor disabilities categorized by a static, nondegenerative encephalopathy arising in the developing brain and associated with deficits in movement, posture, and activity. Spastic CP, which is the most common type, involves high muscle tone and is associated with altered muscle function including poor muscle growth and contracture, increased extracellular matrix deposition, microanatomic disruption, musculoskeletal deformities, weakness, and difficult movement control. These muscle-related manifestations of CP are major causes of progressive debilitation and frequently require intensive surgical and therapeutic intervention to control. Current clinical approaches involve sophisticated consideration of biomechanics, radiologic assessments, and movement analyses, but outcomes remain difficult to predict. There is a need for more precise and personalized approaches involving omics technologies, data science, and advanced analytics. An improved understanding of muscle involvement in spastic CP is needed. Unfortunately, the fundamental mechanisms and molecular pathways contributing to altered muscle function in spastic CP are only partially understood. In this review, we outline evidence supporting the emerging hypothesis that epigenetic phenomena play significant roles in musculoskeletal manifestations of CP.

Ewing’s sarcoma (ES) is caused by a chromosomal translocation leading to the formation of the fused EWSFLI1 gene, which codes for an aberrant transcription factor EWSFLI1. The transcriptional targets of EWSFLI1 have been viewed as promising and novel drug targets in the treatment of ES. One such target is six transmembrane epithelial antigen of the prostate 1 (STEAP1), a transmembrane protein that is upregulated by EWSFLI1 in ES. STEAP1 is a hallmark of tumor invasiveness and an indicator of tumor responsiveness to therapy. EWSFLI1 binds to the STEAP1 promoter region, but the mechanism of action by which it upregulates STEAP1 expression in ES is not entirely understood. Upon analysis of the STEAP1 promoter, we predicted two binding sites for NKX2.2, another crucial transcription factor involved in ES pathogenesis. We confirmed the interaction of NKX2.2 with the STEAP1 promoter using chromatin immunoprecipitation (ChIP) analysis. We used single-molecule RNA imaging, biochemical, and genetic studies to identify the novel role of NKX2.2 in regulating STEAP1 expression in ES. Our results show that NKX2.2 is a co-regulator of STEAP1 expression and functions by interacting with the STEAP1 promoter at sites proximal to the reported EWSFLI1 sites. The co-operative interaction of NKX2.2 with EWSFLI1 in regulating STEAP1 holds potential as a new target for therapeutic interventions for ES.

Spastic type cerebral palsy (CP) is a complex neuromuscular disorder that involves altered skeletal muscle microanatomy and growth, but little is known about the mechanisms contributing to muscle pathophysiology and dysfunction. Traditional genomic approaches have provided limited insight regarding disease onset and severity, but recent epigenomic studies indicate that DNA methylation patterns can be altered in CP. Here, we examined whether a diagnosis of spastic CP is associated with intrinsic DNA methylation differences in myoblasts and myotubes derived from muscle resident stem cell populations (satellite cells; SCs). Twelve subjects were enrolled (6 CP; 6 control) with informed consent/assent. Skeletal muscle biopsies were obtained during orthopedic surgeries, and SCs were isolated and cultured to establish patient–specific myoblast cell lines capable of proliferation and differentiation in culture. DNA methylation analyses indicated significant differences at 525 individual CpG sites in proliferating SC–derived myoblasts (MB) and 1774 CpG sites in differentiating SC–derived myotubes (MT). Of these, 79 CpG sites were common in both culture types. The distribution of differentially methylated 1 Mbp chromosomal segments indicated distinct regional hypo– and hyper–methylation patterns, and significant enrichment of differentially methylated sites on chromosomes 12, 13, 14, 15, 18, and 20. Average methylation load across 2000 bp regions flanking transcriptional start sites was significantly different in 3 genes in MBs, and 10 genes in MTs. SC derived MBs isolated from study participants with spastic CP exhibited fundamental differences in DNA methylation compared to controls at multiple levels of organization that may reveal new targets for studies of mechanisms contributing to muscle dysregulation in spastic CP.

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.

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.