Dottorato in GENETICA E BIOLOGIA MOLECOLARE

Titolo della tesi: Investigating the role of Charme: a chromatin-associated IncRNA supervisor of mammals myogenesis

The mammalian genome contains thousands of long non-coding RNAs (lncRNAs), which have been proposed to be fundamental in the regulation of many biological processes. Among them, nuclear lncRNAs are generally associated to chromatin, where they can act as genome architects contributing to the formation or disassembly of 3D structures. Through a high-throughput transcriptome screening, a subset of new polyadenylated and multi-exonic lncRNAs differentially expressed during murine skeletal muscle differentiation in vitro, was identified (Ballarino et al., 2015). In particular, we focused on one candidate, Charme (for Chromatin architect of muscle expression) that is an abundant and highly conserved non-coding transcript specifically required for in vitro myogenesis. Interestingly, Charme ablation in vivo resulted in a very distinct cardiac pathological phenotype in which the morphology of the murine heart is remodelled (Ballarino et al., 2018). In this work, we deepen the study of the Charme mechanism of action by characterizing its protein interactors. Interestingly, we found that the intron retained Charme isoform (pCharme) is able to specifically bind to MATR3 and PTBP1, two proteins involved in nuclear organization and RNA metabolism. Loss of function experiments demonstrate that PTBP1, supported by MATR3, reinforces the nuclear retention and stabilization of pCharme. Reciprocally, the lncRNA is able to function as a guide for MATR3 recognition of its chromatin targets. Consistent with these data, the targeted deletion of intron 1 by CRISPR/Cas9 in a mouse model, induces the delocalization of the mutant pCharme from the chromatin to the cytoplasm, and results in a muscle phenotype similar to what is observed upon knockout of the full-length transcript (Charme -/-) (Ballarino et al., 2018). Finally, to expand our knowledge on the human Charme (hs-Charme), we generated a CRISPR/CAS9 hs-Charme knock out hiPS cell line and found that the human lncRNA could be involved in the control of cell contraction in cardiomyocytes. Overall, our findings unveil new determinants, which contribute to regulate pCharme activities and reinforce the importance of the intimate interplay between pCharme and the chromatin to allow the lncRNA to exert its function.