Phd in GENETICS AND MOLECULAR BIOLOGY

Thesis title: The regulatory role of long and short RNA crosstalk in motor neuron differentiation

Motor neuronal (MN) identity emerges from a multilayered regulatory architecture in which morphogenetic signaling converges with post-transcriptional processes orchestrated by noncoding RNAs (ncRNAs) and RNA-binding proteins (RBPs). Within this intricate landscape, long noncoding RNAs (lncRNAs) have emerged as versatile molecular players capable of integrating RNA–RNA and RNA–protein networks to fine-tune gene expression. In this thesis, we investigate the molecular and functional features of the human cytoplasmic lncRNA CyCoNP (Cytoplasmic Coordinator of Neural Progenitors), a highly expressed and motor neuron-enriched transcript that sustains neuronal differentiation programs required for the physiology of both neuroblastoma cells and hiPSC-derived MNs. By integrating in silico predictions with endogenous RNA and protein purification, loss-of-function assays, and transcriptomic profiling, we demonstrate that CyCoNP exerts its regulatory activity through RNA-RNA interactions with microRNA-4492 (miR-4492) and NCAM1 mRNA. CyCoNP employs a dual mode of action: in trans, it functions as a miR-4492 sponge by sequestering it from its pro-neuronal targets, including NCAM1 mRNA; in cis, it directly associates with NCAM1 mRNA to modulate the local availability of miR-4492 in its proximity. Furthermore, we describe a direct interaction between CyCoNP and the RBP PTBP1, which modulates miR-4492 binding to the lncRNA and thereby alters its downstream control of NCAM1. Together, these findings expand the understanding of how ncRNAs and RBPs cooperate to shape neuronal gene expression networks, highlighting the relevance of compartmentalized RNA dynamics in the establishment of physiological features.