RNase-based generation of signalling RNAs associated to antiviral immune response

The project aims to contribute to deep into our understanding on the host-virus interplay, with the final goal to design a novel antiviral therapy. Virus infections threaten global health and compromise the viability of our healthcare system. Single-stranded RNA (ssRNA) are highly transmissible viruses, with an elevate risk of zoonosis and potential cause of future pandemia. Among diseases caused by ssRNA viruses we find influenza, which can lead to severe complications, such bronchiolitis, pneumonia or acute respiratory distress syndrome. It is estimated that around 20% of patients affected by viral infections may suffer from organ injury, associated to disabling and high mortality rates. Thus, novel therapies to treat viral infections are among the top priorities of global health policies. One of the key strategies to succeed in the fight against viral infections is the development of innovative solutions that explore alternative mechanism of actions. Interestingly, recent findings highlight the key role of non-coding RNAs (ncRNAs) during the host immune response. Among others, ncRNAs can participate in cell signalling during host response to infection and can be exploited by viruses for the initiation step of replication. Among the key players that determine the content of the small RNA population we find extracellular ribonucleases. Thus, if we want to understand the cell-to-cell RNA talk and not get lost in translation, we must decipher how RNases recognize and shape the small RNA population. In this proposal we aim to identify small ncRNA specifically targeted by the human RNase2, a protein specifically secreted upon ssRNA virus infection. Our Research group has a long-standing expertise in the structural-functional characterization of human secretory RNases. We have determined the proteins specific antipathogen properties and structural determinants for their RNA substrate recognition. Our recent work identified a selective cleavage on tRNA population by RNase2 expressed by macrophages in response to viral RNA. The population of tRNA products generated by specific endonuclease cleavage is emerging as a new information code system that participates in cell defense, adaptation to its environment and cell-to-cell signaling. Interestingly, specific tRNA fragments are proposed as disease biomarkers and even very recently demonstrated as novel therapeutic molecules . In this project we plan to expand our previous studies to an epithelial- macrophage co-culture model infected by the influenza A H1N1 virus. To find out the key tRNA products that act as signalling molecules we plan to perform an integrative coding/ non-coding transcriptomics and epitranscriptomics, using wild-type and RNase2 edited cells. We will also apply high-resolution live cell imaging to visualize the RNase action at the modified RNAs exposed at the cell membrane and its impact in the epithelial barrier viability. The protein-RNA selective recognition will be characterized by an integrative structural analysis and efficacy will be validated first in infected co-cultures and next in a lung organoid model. We are confident that the gained knowledge by the proposed work plan will successfully identify a suitable tRNA fragment candidate to set the basis to develop a future antiviral therapy.