Characterizing Post-transcriptional Modifications of circRNAs to Investigate Biogenesis and Translation.

Recent studies have shown that circular RNAs (circRNAs) are decorated with N6-methyladenosine (mA), 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 mA-dependent regulation of these molecules, we provide protocols that enable circOme-wide detection of mA as well as the perturbation of several components of the mA machinery followed by assays useful to evaluate the impact of their depletion on the production and, when applicable, on the translation of circRNAs.

TDP-43 loss induces extensive cryptic polyadenylation in ALS/FTD.

Nuclear depletion and cytoplasmic aggregation of the RNA-binding protein TDP-43 is the hallmark of ALS, occurring in over 97% of cases. A key consequence of TDP-43 nuclear loss is the de-repression of cryptic exons. Whilst TDP-43 regulated cryptic splicing is increasingly well catalogued, cryptic alternative polyadenylation (APA) events, which define the 3' end of last exons, have been largely overlooked, especially when not associated with novel upstream splice junctions.

CircAFF1 Is a Circular RNA with a Role in Alveolar Rhabdomyosarcoma Cell Migration.

Circular RNAs (circRNAs), covalently closed RNAs that originate from back-splicing events, participate in the control of several processes, including those that occur in the development of pathological conditions such as cancer. Hereby, we describe circAFF1, a circular RNA overexpressed in alveolar rhabdomyosarcoma. Using RH4 and RH30 cell lines, a classical cell line models for alveolar rhabdomyosarcoma, we demonstrated that circAFF1 is a cytoplasmatic circRNA and its depletion impacts cell homeostasis favouring cell migration through the downregulation of genes involved in cell adhesion pathways.

The mA reader YTHDC1 and the RNA helicase DDX5 control the production of rhabdomyosarcoma-enriched circRNAs.

N6-Methyladenosine (mA) 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.

Circular RNA ZNF609/CKAP5 mRNA interaction regulates microtubule dynamics and tumorigenicity.

Circular RNAs (circRNAs) are widely expressed in eukaryotes and are regulated in many biological processes. Although several studies indicate their activity as microRNA (miRNA) and protein sponges, little is known about their ability to directly control mRNA homeostasis. We show that the widely expressed circZNF609 directly interacts with several mRNAs and increases their stability and/or translation by favoring the recruitment of the RNA-binding protein ELAVL1.

CircVAMP3: A circRNA with a Role in Alveolar Rhabdomyosarcoma Cell Cycle Progression.

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 mA pattern opposed to its linear counterpart, suggesting that the two isoforms can be differently regulated by such RNA modification.

Modulation of circRNA Metabolism by mA Modification.

N-methyladenosine (mA) is an RNA modification well-known for its contribution to different processes controlling RNA metabolism, including splicing, stability, and translation of mRNA. Conversely, the role of mA on the biogenesis and function of circular RNAs (circRNAs) has yet to be addressed. circRNAs belong to a class of covalently closed transcripts produced via a back-splicing reaction whereby a downstream 5' splice donor site fuses to an upstream 3' splice acceptor site.

Circ-ZNF609 regulates G1-S progression in rhabdomyosarcoma.

Circular RNAs (circRNAs) represent a class of covalently closed RNAs, derived from non-canonical splicing events, which are expressed in all eukaryotes and often conserved among different species. We previously showed that the circRNA originating from the ZNF609 locus (circ-ZNF609) acts as a crucial regulator of human primary myoblast growth: indeed, the downregulation of the circRNA, and not of its linear counterpart, strongly reduced the proliferation rate of in vitro cultured myoblasts. To deepen our knowledge about circ-ZNF609 role in cell cycle regulation, we studied its expression and function in rhabdomyosarcoma (RMS), a pediatric skeletal muscle malignancy.