Phd in GENETICS AND MOLECULAR BIOLOGY

Thesis title: Investigating the function of circ-Dlc1 in vitro and in vivo

The work presented in this thesis focuses on circ-Dlc1, a conserved FUS-dependent circRNA that was firstly identified and found to be highly expressed in murine in vitro-derived motor neurons (MNs). CircRNAs are covalently-closed circular molecules which arise following an alternative splicing event termed backsplicing. To gain insight into the function of circ-Dlc1, a knock-out (KO) mouse model was generated by employing a CRISPR/Cas9 strategy designed to target the intronic sequences responsible for its biogenesis. A primary phenotypic screening comprised the treadmill exhaustion test to assess the presence of neuromuscular defects, as circ-Dlc1 is expressed in MNs and mutations in FUS are known to be associated with inherited forms of Amyotrophic Lateral Sclerosis (ALS). Indeed, the KO mice present clear defects in the proper completion of the test compared to syngeneic WT animals. The presence of circ-Dlc1 was then explored in different districts of the brain via qRT-PCR; its expression was found to be highest in the cortex and the striatum, indicating that the observed phenotype might be due to alterations in central-cortical functions. This was confirmed by RNA-FISH using a new single-molecule technology: circ-Dlc1 preferentially localises in deeper layers of the prefrontal cortex containing distant motor structure-projecting neurons that control movement. In addition, proteomic analyses of the striatum revealed deregulations in glutamatergic-associated circuitries including long-term potentiation (LTP). Pulldown experiments performed using tissue-derived extracts are ongoing for the identification of circ-Dlc1-interacting molecules, in order to shed light on a putative mechanism of action. One of these interactors, miR-130b-5p, was detected in the nucleus of cells expressing striatal and pyramidal tract neuronal marker Ctip2, even more so in cells lacking the expression of the circRNA. An interesting speculation could point towards the circ-Dlc1-miR-130b-5p interaction being responsible for the nucleocytoplasmic translocation/sequestering of the miRNA and consequent regulation of said LTP-associated protein levels at the synapse, contributing to alterations in corticostriatal connectivity.