Thesis title: Functional characterization of the motor neuron-enriched circHdgfrp3(2,3,4,5) RNA in physiological and pathological conditions
Circular RNAs (circRNAs) are a class of covalently closed transcripts resulting from a non-canonical splicing reaction, known as back-splicing, that have recently sparked growing attention. In fact, an increasing number of circRNAs have been found to regulate gene expression, while also being involved in different physiological and pathological processes. CircRNAs are evolutionary conserved and display tissue- and time- specific expression patterns, being especially enriched in the brain during development and neurogenesis. Intriguingly, neuronal circRNAs display dynamic localization at synapses and neurites, suggesting they could exert roles relevant for neuronal activity. This thesis focuses on the functional characterization of circHdgfrp3(2,3,4,5), a conserved circRNA whose biogenesis is controlled by the FUS protein and that is up-regulated in murine motor neurons (MNs). Firstly, circHdgfrp3(2,3,4,5) was found to localize in neurites during MN maturation by employing a sensitive single-molecule FISH (smFISH) approach. We then found that circHdgfrp3(2,3,4,5) may traffic among neurites but that this ability is impaired by oxidative stress, a condition that causes the perinuclear retention of the circRNA in stress granules (SGs) and a consequent reduction of its presence in neurites. In MNs carrying an ALS-linked FUS mutation, a higher fraction of circHdgfrp3(2,3,4,5) was trapped in both SGs and additional cytoplasmic FUS-aggregates upon oxidative stress conditions. Notably, upon stress removal circHdgfrp3(2,3,4,5) was easily released from SGs and its peripheral localization was restored. However, the release of circHdgfrp3(2,3,4,5) from FUS-aggregates was proven to be less efficient. Altogether, these results suggest that the long-lasting trapping of circHdgfrp3(2,3,4,5) in FUS-aggregates in ALS-related conditions might interfere with its trafficking as well as with its functional role. In addition, six mRNAs were identified as circHdgfrp3(2,3,4,5) interacting partners by performing a psoralen (AMT)-cross-linked RNA pull-down followed by RNA-sequencing (RNA-seq) analysis in MNs. Interestingly, the localization of one of these mRNA interactors, Eif3c, was affected upon circHdgfrp3(2,3,4,5) knock-down in oxidative stress conditions. Furthermore, circHdgfrp3(2,3,4,5) and several of its RNA interactors were shown to be enriched at motile organelles such as at endosomes and lysosomes by performing APEX-catalyzed in situ proximity labeling in living cells. Moreover, the association of circHdgfrp3(2,3,4,5) mRNA interactors with late endosomes was revealed to decrease upon the circRNA knock-down. These data point towards a possible involvement of circHdgfrp3(2,3,4,5) in properly localizing its RNA binding partners near endosomes to either promote their local translation or their transport towards peripheral neuronal districts. Overall, the work presented herein provides new insights into the possible role played by circHdgfrp3(2,3,4,5) in MNs by shedding light on its sub-cellular localization and function, whose deregulation may be linked to neurodegenerative pathways.