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MUNCODD Report Summary

Project ID: 340172
Funded under: FP7-IDEAS-ERC
Country: Italy

Mid-Term Report Summary - MUNCODD (Role of long non coding RNA in muscle differentiation and disease)

The field of interest applies to the study of the molecular networks controlling normal muscle homeostasis and to identify the alterations occurring in pathological conditions. In particular, it aims at identifying the role played in these processes by new classes of non canonical RNAs: the long non coding RNAs (lncRNAs) and the circular RNAs (circRNAs). While the function of well established master regulators, such as transcriptional factors and miRNAs, has been largely defined much less is known about the role of this previously disregarded class of transcripts. Therefore, a major objective of this project is to re-evaluate and re-design molecular circuitries controlling myogenesis and, in more general terms, to shed light on the biogenesis and function of this new and complex class of transcripts and how they contribute to cellular and organismal biology. This new field of research should also constitute a vast and largely unexplored territory for the development of novel therapeutics and diagnostics.
The major findings can be summarized in the following points:
1. Identification of several novel circuitries of gene expression regulation for lncRNAs
In particular, Charme, a chromatin-associated nuclear lncRNA highly conserved between mouse and human, was identified as a species controlling the expression of several myogenic genes; among them, genes implicated in several types of cardiomyopathies. Notably, Charme depletion in mice produced a very clear cardiac hyperthrophic phenotype. Using molecular and imaging methodologies, we were able to show that Charme contributes to chromosome architecture by bringing into close proximity genomic regions which are more than 90 Mb apart and which contain genes that are coordinately expressed. Therefore, Charme provides an important and unprecedented example of a chromatin associated lncRNA acting in controlling chromatin architecture and in organizing functional transcriptional activating domains (TADs) where coordinated gene expression occurs. Moreover, the strong impact on heart morphology shows that such activity has a considerable impact in vivo on tissue homeostasis.
2. Identification of circRNAs with a relevant function in myogenesis
From RNAseq data of murine and human myoblasts and myotubes we were able to identify a numerous class of circRNAs differentially expressed during differentiation and highly conserved in both species. A high-content functional genomic screen, using modified siRNAs specifically designed for circRNA knockdown, was applied to the study of human myoblast differentiation. Several interesting phenotypes were obtained. In particular, the depletion of circ-ZNF609, had a strong impact on myoblast proliferation. circ-ZNF609 was analysed in more detail due to the interesting finding that it contains a potential open reading frame. Several experiments proved the coding nature of circ-ZNF609 and indicated that it is indeed translated through a novel regulatory pathway, which responds to cellular stress. Therefore, circ-ZNF609 provides the first example of a protein-coding circRNA in eukaryotes and offers interesting material for the study of new pathways of cellular translation that are activated in response to environmental changes.
3. Identification of a potential new therapeutic approach for Duchenne Muscular Dystrophy (DMD)
Among several DMD patients under analysis, we identified a boy (GSdelta44) with a Duchenne genotype (deletion of exon 44) who instead displayed an almost asymptomatic phenotype (Becker type). We discovered that this individual produces a natural skipping of exon 45 that partially rescues dystrophin synthesis. By studying his transcriptome we found that this is due to the lack of a specific protein, the splicing factor Clef2a, which in normal conditions enhances the inclusion of exon 45. Several experiments demonstrated the central role of Celf2a in controlling exon 45 splicing, not lastly the fact that Celf2a interacts in vivo with exon 45 of the dystrophin pre-mRNA. These data suggested that inhibition of this factor can become a powerful new therapeutic approach for those DMD cases where skipping of this exon could be of therapeutic benefit or even represent a strategy for improving other DMD therapeutic treatments. In vitro experiments have indeed shown that depletion of this factor is curative for DMD myoblasts with exon 44 deletion. Experiments are ongoing to identify compounds that could either repress the activity of the Celf2a gene or inhibit protein function.

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