The discovery of introns over four decades ago revealed a new vision of genes and their interrupted arrangement. Throughout the years, it has appeared that introns play essential roles in the regulation of gene expression. Unique processing of excised introns through the formation of lariats suggests a widespread role for these molecules in the structure and function of cells. In addition to rapid destruction, these lariats may linger on in the nucleus or may even be exported to the cytoplasm, where they remain stable circular RNAs (circRNAs). Alternative splicing (AS) is a source of diversity in mature transcripts harboring retained introns (RI-mRNAs). Such RNAs may contain one or more entire retained intron(s) (RIs), but they may also have intron fragments resulting from sequential excision of smaller subfragments via recursive splicing (RS), which is characteristic of long introns. There are many potential fates of RI-mRNAs, including their downregulation via nuclear and cytoplasmic surveillance systems and the generation of new protein isoforms with potentially different functions. Various reports have linked the presence of such unprocessed transcripts in mammals to important roles in normal development and in disease-related conditions. In certain human neurological-neuromuscular disorders, including myotonic dystrophy type 2 (DM2), frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS) and Duchenne muscular dystrophy (DMD), peculiar processing of long introns has been identified and is associated with their pathogenic effects. In this review, we discuss different mechanisms involved in the processing of introns during AS and the functions of these large sections of the genome in our biology.

Expandable (CTG)n repeats in the 3′ UTR of the DMPK gene are a cause of myotonic dystrophy type 1 (DM1), which leads to a toxic RNA gain-of-function disease. Mutant RNAs with expanded CUG repeats are retained in the nucleus and aggregate in discrete inclusions. These foci sequester splicing factors of the MBNL family and trigger upregulation of the CUGBP family of proteins resulting in the mis-splicing of their target transcripts. To date, many efforts to develop novel therapeutic strategies have been focused on disrupting the toxic nuclear foci and correcting aberrant alternative splicing via targeting mutant CUG repeats RNA; however, no effective treatment for DM1 is currently available. Herein, we present results of culturing of human DM1 myoblasts and fibroblasts with two small-molecule ATP-binding site-specific kinase inhibitors, C16 and C51, which resulted in the alleviation of the dominant-negative effects of CUG repeat expansion. Reversal of the DM1 molecular phenotype includes a reduction of the ...

Circular RNAs (circRNAs) are a class of single-stranded covalently closed RNA rings. Biogenesis of circRNAs, which may occur co-transcriptionally and post-transcriptionally via a back-splicing mechanism, requires the presence of complementary and/or inverted repeat sequences in introns flanking back-spliced exons and is facilitated by RNA-binding proteins. CircRNAs are abundant across eukaryotes; however, their biological functions remain largely speculative. Recently, they have been emerging as new members of a gene regulatory network and contributing factors in various human diseases including cancer, neurological, muscular and cardiovascular disorders. In this review, we present an overview of the current knowledge about circRNAs biogenesis and their aberrant expression in various human disorders. In particular, we focus on the latest discovery of circRNAs global upregulation in myotonic dystrophy type 1 (DM1) skeletal muscles and the role these prospective biomarkers might have for prognosis and therapeutic response in DM1.

Myotonic dystrophy type 1 (DM1) is an RNA-based disease with no current treatment. It is caused by a transcribed CTG repeat expansion within the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Mutant repeat expansion transcripts remain in the nuclei of patients’ cells, forming distinct microscopically detectable foci that contribute substantially to the pathophysiology of the condition. Here, we report small-molecule inhibitors that remove nuclear foci and have beneficial effects in the HSA^LR mouse model, reducing transgene expression, leading to improvements in myotonia, splicing, and centralized nuclei. Using chemoproteomics in combination with cell-based assays, we identify cyclin-dependent kinase 12 (CDK12) as a druggable target for this condition. CDK12 is a protein elevated in DM1 cell lines and patient muscle biopsies, and our results showed that its inhibition led to reduced expression of repeat expansion RNA. Some of the inhibitors identified in this study are currently the subject of clinical trials for other indications and provide valuable starting points for a drug development program in DM1.

Myotonic dystrophy type 1 (DM1) is a progressive, multisystemic disorder caused by an expansion of CTG repeats in the 3’ untranslated region of the DMPK gene. When transcribed the mutant RNAs accumulate in affected tissues appearing as distinct foci when visualised by in situ hybridisation. The RNA foci are aggregates of CUG repeat-containing RNAs that sequester RNA-binding proteins, particularly muscleblind-like (MBNL) proteins, leading to their dysfunction and causing downstream molecular and cellular defects. Here we show the double knock-out of MBNL1 and 2 prevents RNA foci formation and nuclear retention of mutant DMPK mRNA in DM1 cells as well as promoting their degradation and nuclear export. Using stochastic optical reconstruction microscopy (STORM), we find the presence of both large foci and micro foci in DM1 cells. Large foci consist of multiple DMPK transcripts, while many micro foci are (CUG)n fragments. The absence of MBNL proteins not only prevents the aggregation of multiple DMPK transcripts into large foci, but also promotes their degradation and nuclear processing. However, although a substantial amount of MBNL1 proteins are bound to the mutant transcripts, the pools of free MBNL1 proteins are similar in DM1 nuclei to those in controls. Furthermore, we have identified several factors that are involved in the control of mutant DMPK mRNA turnover, including XRN2, EXOSC10, UPF1 and STAU1. Our study indicates that these factors are implicated in the RNA foci accumulation and the degradation of mutant DMPK mRNA. UPF1 and STAU1 may have additional roles beyond degradation, impacting the nuclear processing of mutant DMPK mRNA. Our study also highlights the critical role of MBNL proteins in regulating mutant DMPK mRNA metabolism: the absence of MBNLs in DM1 appears to expedite the processing of mutant DMPK mRNA mediated by these RNA decay factors. Significance statement: Our investigations uncovered valuable data on the RNA foci dynamics in DM1, revealing the intricate mechanisms that underlie their formation, stability, and turnover. Our findings also contributed to delineate the complex pathways involved in the transportation and degradation of the mutant mRNA and provided insights into the critical role played by MBNL proteins in these processes. Studying the degradation mechanism of mutant DMPK mRNA in myotonic dystrophy may provide a foundation for comprehending the mechanisms of RNA degradation in other diseases caused by short tandem repeat (STR) mutations, such as Huntington’s disease, Fragile X syndrome, and several types of ataxia. Additionally, the use of cutting-edge STORM technology can provide a valuable tool for investigating RNA foci in other STR expansion disorders.

Splicing aberrations induced as a consequence of the sequestration of MBNL splicing factors on the DMPK transcript, which contains expanded CUG repeats, present a major pathomechanism of myotonic dystrophy type 1 (DM1). As MBNLs 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 twenty 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. Author Summary: Recently, a great deal of interest has been focused on a new class of RNA molecules called circular RNAs (circRNAs). To date, thousands of circRNAs have been found in different human cells/tissues. Although the function of circRNAs remains mostly unknown, circRNAs have emerged as an important component of the RNA-RNA and RNA-protein interactome. Thus, intensive efforts are being made to fully understand the biology and function of circRNAs, especially their role in human diseases. As an important role in the biogenesis of circRNA may be played by MBNL splicing factors, in this study we used DM1 (to a lesser extent, DM2) as a natural model in which the level of MBNLs is decreased. In contrast to the expected effect, our results consistently showed a global increase in circRNA levels in DM1. As a consequence, whole genome transcriptome analysis revealed dozens of circRNAs with significantly altered (mostly increased) levels in DM1. Furthermore, we observed that the circRNA levels were in many cases strongly associated with DM1 severity.