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. We found that circ-ZNF609 is upregulated in biopsies from the two major RMS subtypes, embryonal (ERMS) and alveolar (ARMS). Moreover, we discovered that in an ERMS-derived cell line circ-ZNF609 knock-down induced a specific block at the G1-S transition, a strong decrease of p-Akt protein level and an alteration of the pRb/Rb ratio. Regarding p-Akt, we were able to show that circ-ZNF609 acts by counteracting p-Akt proteasome-dependent degradation, thus working as a new regulator of cell proliferation-related pathways. As opposed to ERMS-derived cells, the circRNA depletion had no cell cycle effects in ARMS-derived cells. Since in these cells the p53 gene resulted downregulated, with a concomitant upregulation of its cell cycle-related target genes, we suggest that this could account for the lack of circ-ZNF609 effect in ARMS.

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.

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. We developed a novel bioinformatic approach to reliably identify distinct APA event types: alternative last exons (ALE), 3’UTR extensions (3’Ext) and intronic polyadenylation (IPA) events. We identified novel neuronal cryptic APA sites induced by TDP-43 loss of function by systematically applying our pipeline to a compendium of publicly available and in house datasets. We find that TDP-43 binding sites and target motifs are enriched at these cryptic events and that TDP-43 can have both repressive and enhancing action on APA. Importantly, all categories of cryptic APA can also be identified in ALS and FTD post mortem brain regions with TDP-43 proteinopathy underlining their potential disease relevance. RNA-seq and Ribo-seq analyses indicate that distinct cryptic APA categories have different downstream effects on transcript and translation. Intriguingly, cryptic 3’Exts occur in multiple transcription factors, such as ELK1 , SIX3, and TLX1, and lead to an increase in wild-type protein levels and function. Finally, we show that an increase in RNA stability leading to a higher cytoplasmic localisation underlies these observations. In summary, we demonstrate that TDP-43 nuclear depletion induces a novel category of cryptic RNA processing events and we expand the palette of TDP-43 loss consequences by showing this can also lead to an increase in normal protein translation.

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.

Cryptic splicing caused by TDP-43 proteinopathy is a hallmark of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, which cryptic splicing events (CEs) are the most sensitive to TDP-43 depletion, where CEs localise within cells, and how specific CEs are in human tissues is poorly defined. Analyses of in vitro TDP-43 knockdowns and postmortem RNA-seq datasets revealed that a small subset out of thousands of CEs are specific markers for TDP-43 proteinopathy in vivo . Nonsense-mediated decay (NMD) masked a portion of CEs, influencing their subcellular localization and detectability in tissue. Dose-dependent TDP-43 depletion identified “early-responsive” CEs, which possess stronger splice sites and denser, more canonical TDP-43 binding motifs. Finally, we developed a composite cryptic burden score that effectively captured TDP-43 pathology across heterogeneous tissues and correlated with regional vulnerability and genetic background. Our work identifies robust biomarkers and offers new insights into TDP-43-mediated splicing dysregulation in neurodegeneration.

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 presented data underline the importance of this circular RNA as a new partial suppressor of the alveolar rhabdomyosarcoma tumour progression and as a putative future therapeutic target.