Dottorato in GENETICA E BIOLOGIA MOLECOLARE

Titolo della tesi: The long non-coding RNA lncMN2 functions as a competing endogenous RNA in motor neuron differentiation

Long non-coding RNAs (lncRNAs) are currently recognized as crucial players in nervous system development, function and pathology. In this study we used an in-vitro differentiation system to generate mouse motor neurons (MNs) from embryonic stem cells and we analyzed, via RNA-Seq, the long non-coding transcriptome of MNs. We selected 12 species of lncRNAs based on their expression levels and fold change along MN differentiation. Among these candidates, I focused my attention on lncRNA 5330434G04Rik (named lncMN2) highlighting peculiar structural and localization features. Functional characterization of the selected lncRNA was performed through CRISPR/Cas9 – KO approach. Cytofluorimetric and phenotypical analysis reveal an impaired motor neuronal differentiation in the lncMN2 – KO clones. Moreover, bioinformatics predictions highlighted a 100nt region on the lncRNA able to bind miR-466i, afterwards confirmed by CLIP and reporter assays, imputing a role to lncMN2 as a ceRNA. Functional characterization of the miRNA binding region was performed through CRISPR/Cas9 – KO approach. Cytofluorimetric and phenotypical analysis on mir-466i binding region – KO clones resembled the already-observed lncMN2-KO phenotypes. Putative miRNA targeted transcripts, underlying the phenotypical evidences, were highlighted through scRNA-seq and confirmed through molecular analysis. Our work aims to investigate lncRNA functions in motor neuronal commitment pathways and is a step toward the comprehension of the still poorly-characterized ceRNA mechanisms in motoneuronal circuitries.