Transcriptome-wide profiles of circular RNA and RNA-binding protein interactions reveal effects on circular RNA biogenesis and cancer pathway expression

Abstract

Background Circular RNAs (circRNAs) are stable, often highly expressed RNA transcripts with potential to modulate other regulatory RNAs. A few circRNAs have been shown to bind RNA-binding proteins (RBPs); however, little is known about the prevalence and distribution of these interactions in different biological contexts. Methods We conduct an extensive screen of circRNA-RBP interactions in the ENCODE cell lines HepG2 and K562. We profile circRNAs in deep-sequenced total RNA samples and analyze circRNA-RBP interactions using a large set of eCLIP data with binding sites of 150 RBPs. We validate interactions for select circRNAs and RBPs by performing RNA immunoprecipitation and functionally characterize our most interesting candidates by conducting knockdown studies followed by RNA-Seq. Results We generate a comprehensive catalog of circRNA-RBP interactions in HepG2 and K562 cells. We show that KHSRP binding sites are enriched in flanking introns of circRNAs and that KHSRP depletion affects circRNA biogenesis. We identify circRNAs that are highly covered by RBP binding sites and experimentally validate individual circRNA-RBP interactions. We show that circCDYL, a highly expressed circRNA with clinical and functional implications in bladder cancer, is almost completely covered with GRWD1 binding sites in HepG2 cells, and that circCDYL depletion counteracts the effect of GRWD1 depletion. Furthermore, we confirm interactions between circCDYL and RBPs in bladder cancer cells and demonstrate that circCDYL depletion affects hallmarks of cancer and perturbs the expression of key cancer genes, e.g., TP53. Finally, we show that elevated levels of circCDYL are associated with overall survival of bladder cancer patients. Conclusions Our study demonstrates transcriptome-wide and cell-type-specific circRNA-RBP interactions that could play important regulatory roles in tumorigenesis.

Circular RNA circRHOBTB3 represses metastasis by regulating the HuR-mediated mRNA stability of PTBP1 in colorectal cancer

“…However, the patterns of circRNA-protein interactions are more complex and interesting than those of circRNA-miRNA interactions and have been the subject of great focus recently 50 . RBPs affect all phases of the circRNA lifecycle, including circRNA biogenesis, localization, function and degradation 51 . Herein, we determined that increased expression of circRHOBTB3 restrains PTBP1 expression by binding to HuR and facilitating its ubiquitination and degradation, which consequently restrains CRC metastasis.…”

Circular RNAs as Novel Regulators of β-Cell Functions under Physiological and Pathological Conditions

“…Recently, an extensive screening of circRNA-RBP interactions has been performed, with the aim of obtaining a broader picture of the circRNA-RBP interactome [ 58 ]. A comprehensive catalogue of circRNA-RBP interactions in HepG2 and K562 cells was generated, and several candidates have been characterized [ 58 ]. In particular, it was demonstrated that circCDYL associates to IGF2BP1 and IGF2BP2, and that this interaction modulates proliferation of bladder cancer cells.…”

“…In particular, it was demonstrated that circCDYL associates to IGF2BP1 and IGF2BP2, and that this interaction modulates proliferation of bladder cancer cells. Indeed, the inhibition of circCDYL perturbs the expression of key cancer genes, and elevated levels of circCDYL are linked to the overall survival of bladder cancer patients [ 58 ].…”

Circular RNA: A novel type of biomarker for glioma (Review)

“…Notably, numerous circRNAs that do not possess miRNA-binding sites still have a wide range of biological functions, indicating that circRNAs have their own additional regulatory functions. Previous studies have revealed that circRNAs may interact with RBPs or other proteins to form circRNA-protein complexes, which may impact the function and mechanisms of action of binding proteins ( 62 , 63 ). For example, circ-FOKX2 may integrate with Y-box binding protein 1 and heterogeneous nuclear ribonucleoprotein K to facilitate the upregulation of the oncogenes NUF2 and pyridoxal kinase, ultimately accelerating pancreatic ductal adenocarcinoma progression ( 64 ).…”