Circular RNA Primer

Cells contain a striking diversity of RNA types, many of which have been implicated in the pathogenesis of neuromuscular disease. Unlike most RNAs, circular RNAs (circRNAs) are single-stranded, covalently closed loops. CircRNAs arise via one of three mechanisms: (a) direct ligation of 5′ and 3′ ends of linear RNAs, (b) as intermediates in RNA processing reactions, or (c) via “back splicing,” when a downstream 5′ splice site (donor) is joined to an upstream 3′ splice site (acceptor). A variety of biologic roles for circRNAs have been identified.

Presence of circRNA in DM1 Skeletal Muscle

While aberrant RNA splicing represents a central disease mechanism in DM1, virtually nothing is known regarding the potential for dysregulation of circRNAs. Dr. Fabio Martelli (IRCCS Policlinico San Donato) and colleagues have recently published an analysis of dysregulation of circRNAs in DM1 patient skeletal muscle biopsies (Voellenkle et al., 2019). The authors show that specific myogenesis-associated circRNAs are altered in DM1 biopsies and in DM1 patient myogenic cell cultures.

The research team identified specific circRNAs through review of 30 published DM1 RNAseq databases—relative abundance of a circRNA to its linear counterpart was used as an initial filter, followed by comparison to a list of circRNAs that were previously identified in human or murine myoblasts. Thus, the analysis was not comprehensive, but geared toward identification of transcripts most likely to be dysregulated in skeletal muscle tissue. Candidate circRNAs meeting the investigators’ criteria then were validated using qPCR of skeletal muscles from DM1 subjects and age-/sex-matched controls. Primer specificity was confirmed and the possibility that results were due to a general increase in transcription in DM1 was excluded, and results were confirmed in independent muscle biopsies.

Taken together, four circRNAs—circCDYL, circHIPK3, circRTN4_03, and circZNF609—exhibited significantly increased circular-to-linear RNA ratio in DM1 muscles versus controls. The research team subsequently used receiver operating characteristic curve analysis and confirmed that a transcript’s circular-to-linear ratio could discriminate between DM1 and healthy controls. Finally, the increase in circular fraction for the four circRNAs correlated with a variety of clinical and molecular characteristics of study subjects. Circular fraction ratios correlated with both skeletal muscle strength and splicing biomarkers of disease severity.  Moreover, circular fraction was higher in the more severely affected DM1 patients. Induction of two of the dysregulated circRNAs (circCDYL and circRTN4) was also detected in plasma. Finally, analyses of DM1 myogenic cell lines identified a pattern of circRNA dysregulation that was, in part, similar to data obtained in patient muscle biopsies.

Potential Utility of Dysregulated circRNAs in DM1

The research team self-identified caveats and described these findings as pilot data. Any putative contributing role that dysregulated circRNAs may have in the pathogenesis of DM1 is currently unknown. Yet the discovery of specific, dysregulated circRNAs in DM1 skeletal muscle, if confirmed, may offer advantages for drug development efforts—circRNAs are exceptionally stable in that they are resistant to exonuclease degradation and their dysregulation was detectable in plasma and myogenic cell lines from DM1 patients. These traits make them amenable to use as pharmacodynamic biomarkers for clinical studies and trials in DM1.

Reference:

Dysregulation of Circular RNAs in Myotonic Dystrophy Type 1.
Voellenkle C, Perfetti A, Carrara M, Fuschi P, Renna LV, Longo M, Sain SB, Cardani R, Valaperta R, Silvestri G, Legnini I, Bozzoni I, Furling D, Gaetano C, Falcone G, Meola G, Martelli F.
Int J Mol Sci. 2019 Apr 19;20(8). pii: E1938. doi: 10.3390/ijms20081938.