Transcriptome-wide profiles of circular RNA and RNA-binding protein interactions reveal effects on circular RNA biogenes
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RESEARCH
Open Access
Transcriptome-wide profiles of circular RNA and RNA-binding protein interactions reveal effects on circular RNA biogenesis and cancer pathway expression Trine Line Hauge Okholm1,2* , Shashank Sathe3, Samuel S. Park3, Andreas Bjerregaard Kamstrup4, Asta Mannstaedt Rasmussen1,2, Archana Shankar3, Zong Ming Chua3, Niels Fristrup5, Morten Muhlig Nielsen1,2, Søren Vang1,2, Lars Dyrskjøt1,2, Stefan Aigner3, Christian Kroun Damgaard4, Gene W. Yeo3 and Jakob Skou Pedersen1,2,6*
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. Keywords: Circular RNAs, RNA-binding proteins, RBP sponges, circCDYL, Bladder cancer, Tumorigenesis
* Correspondence: [email protected]; [email protected] 1 Department of Molecular Medicine (MOMA), Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the so
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