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N6-Methyladenosine (m6A) is well-known for controlling different processes of linear RNA metabolism. Conversely, its role in the biogenesis and function of circular RNAs (circRNAs) is still poorly understood. Here, we characterize circRNA expression in the pathological context of rhabdomyosarcoma (RMS), observing a global increase when compared to wild-type myoblasts. For a set of circRNAs, such an increase is due to the raised expression of the m6A machinery, which we also find to control the proliferation activity of RMS cells. Furthermore, we identify the RNA helicase DDX5 as a mediator of the back-splicing reaction and as a co-factor of the m6A regulatory network. DDX5 and the m6A reader YTHDC1 are shown to interact and to promote the production of a common subset of circRNAs in RMS. In line with the observation that YTHDC1/DDX5 depletion reduces RMS proliferation, our results provide proteins and RNA candidates for the study of rhabdomyosarcoma tumorigenicity. Rhabdomyosarcoma (RMS) is the most diffused soft tissue sarcoma in children and adolescents. Herein, the authors identify the m6A machinery and the RNA helicase DDX5 as factors responsible for the increase of a subset of circRNAs in RMS, providing protein and RNA candidates for the study of its tumorigenicity. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs), a class of covalently closed RNAs formed by a back-splicing reaction, have been involved in the regulation of diverse oncogenic processes. In this article we describe circVAMP3, a novel circular RNA overexpressed in RH4, a representative cell line of alveolar rhabdomyosarcoma. We demonstrated that circVAMP3 has a differential m6A pattern opposed to its linear counterpart, suggesting that the two isoforms can be differently regulated by such RNA modification. Moreover, we show how circVAMP3 depletion in alveolar rhabdomyosarcoma cells can impair cell cycle progression, through the alteration of the AKT-related pathways, pointing to this non-coding RNA as a novel regulator of the alveolar rhabdomyosarcoma progression and as a putative future therapeutic target. [ABSTRACT FROM AUTHOR]

Recent studies have shown that circular RNAs (circRNAs) are decorated with N6-methyladenosine (m 6 A), a co-transcriptional modification known to participate in the regulation of many processes governing linear RNA metabolism. Nevertheless, the activity of this mark on circRNAs is still poorly understood. In order to facilitate the study of m 6 A-dependent regulation of these molecules, we provide protocols that enable circOme-wide detection of m 6 A as well as the perturbation of several components of the m 6 A machinery followed by assays useful to evaluate the impact of their depletion on the production and, when applicable, on the translation of circRNAs. Other modifications exist and can be explored following the same principles., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)