Global Increase in Circular RNA Levels in Myotonic Dystrophy

Abstract

Splicing aberrations induced as a consequence of the sequestration of muscleblind-like splicing factors on the dystrophia myotonica protein kinase transcript, which contains expanded CUG repeats, present a major pathomechanism of myotonic dystrophy type 1 (DM1). As muscleblind-like factors may also be important factors involved in the biogenesis of circular RNAs (circRNAs), we hypothesized that the level of circRNAs would be decreased in DM1. To test this hypothesis, we selected 20 well-validated circRNAs and analyzed their levels in several experimental systems (e.g., cell lines, DM muscle tissues, and a mouse model of DM1) using droplet digital PCR assays. We also explored the global level of circRNAs using two RNA-Seq datasets of DM1 muscle samples. Contrary to our original hypothesis, our results consistently showed a global increase in circRNA levels in DM1, and we identified numerous circRNAs that were increased in DM1. We also identified many genes (including muscle-specific genes) giving rise to numerous (>10) circRNAs. Thus, this study is the first to show an increase in global circRNA levels in DM1. We also provided preliminary results showing the association of circRNA level with muscle weakness and alternative splicing changes that are biomarkers of DM1 severity.

An Overview of Circular RNAs and Their Implications in Myotonic Dystrophy

“…The involvement of RBP20 in biogenesis of these TTN circRNAs was confirmed in the Rbm20 null mice that completely lacked these molecules, and in a cardiac sample from a heterozygous RBM20 mutation carrier in which production of TTN circRNAs was severely affected [9]. Interestingly, a high number of circRNAs (~100) originated predominantly from the I-band of TTN was also detected in human skeletal muscles from DM1 patients as reported most recently by Czubak et al, [40].…”

“…Interestingly, the elevation of Mbl was found to increase not only circMbl, but also the levels of several other circRNAs, suggesting a more general role of the protein in circRNA biogenesis. However, most recent results from two research groups [36,40], which used DM1 muscle biopsy tissues as a natural model of MBNLs deficiency, showed instead a global elevation of circRNA levels. This discovery questions the original idea of MBNLs being universal factors of circRNAs biogenesis, and suggests that the proteins, rather than playing a global regulatory role, facilitate the formation of individual species of circRNAs.…”

“…Recent rapid interest in this class of molecules has resulted in considering circRNAs as a group of physiological RNA molecules with an important role in normal cell biology, and circRNAs dysregulation has been reported to be associated with human diseases. The expression of circRNAs has recently been reported to generally decrease in human cancers [33,34,35] and increase in some late-onset human neurological and muscular disorders [9,15,36,37,38,39,40] suggesting that their expression can be drastically altered and potentially contribute to pathological states. There are reports suggesting circRNA involvement in cardiovascular and autoimmune conditions [41,42,43,44,45,46].…”

“…Notably, circRNA abundance tends to increase during the differentiation process, and they were found to be highly expressed in mammalian brain and striated muscle tissues (both skeletal and cardiac) and they are one of the body tissues showing the highest levels of alternative splicing. Lately, an elevated pattern of expression of circRNAs was documented in skeletal muscles of patients suffering from Duchenne muscular dystrophy (DMD) and myotonic dystrophy type 1 (DM1) [36,40,47,48]. In both the diseases the differential expression of circRNAs levels was correlated with aberrant alternative splicing.…”

“…For example, mis-splicing of the CLCN1 exon 7, the INSR exon 11, and the BIN1 exon 11 were shown to be associated with reduced chloride conductance, lower insulin responsiveness, and muscle weakness, respectively [54,55,56,57]. However, nuclear deposition of rCUGexp leads to abnormalities in many pathways of RNA metabolism [58] and recently discovered dysregulation of circRNAs in DM1 muscles may be one of them [36,40].…”