Thesis title: RNAs in cell identity shaping: from lncRNAs in Motoneurons to mRNA metabolism in Rhabdomyosarcoma
During my Ph.D., I had the opportunity to work on two distinct research projects that, although different in terms of topic, systems adopted and methodologies employed, share a common denominator: the RNA.
The incredible versatility of RNA has prompted it to occupy a prominent position in the regulation of gene expression. Indeed, on the one hand, evolution has encouraged the expansion of the noncoding genome to constitute 98% of the human genetic material, thus providing a wide range of regulatory elements and noncoding RNAs. On the other hand, the development of RNA processing mechanisms, such as splicing and polyadenylation, has considerably extended the functional repertoire of the remaining 2% of the coding genome so that a large number of RNAs can be obtained from a single gene. This evidence has prompted a shift away from the traditional protein-centric perspective of gene expression towards a greater emphasis on the role of RNA.
The first project, the primary focus of this thesis, aimed to analyze the function of Lhx1os, a long non-coding RNA (lncRNA) specific to motoneurons, characterized by increased expression in both human and mouse models of ALS.
We observed that knockdown of Lhx1os in mice resulted in impaired motor function and a decrease in the number of mature motoneurons in the spinal cord. In addition, we identified the specific alteration of the ER stress-response pathway, with the downregulation of Unfolded Protein Response (UPR) factors.
We proceeded to elucidate the mechanism of action of Lhx1os through the identification of its interactors. First, we observed that Lhx1os binds to ER-associated PDIA3 disulfide isomerase, and that both factors affect the expression of the same set of genes, suggesting their cooperation in the regulation of the UPR. From the other, we discovered the presence of a Transposable Element (TE) within the Lhx1os sequence, responsible for the interaction with a set of mRNAs encoding proteins involved in ER-stress.
Overall, the observed phenotype and function indicate the significant role exerted by Lhx1os in the control of motoneuron homeostasis and health.
The second project, currently ongoing, aims to shed light on the role of the m6A reader YTHDC1 and the RNA helicase DDX5 in the tumor processes of rhabdomyosarcoma (RMS).
With a particular look at their contribution to RNA metabolism through the control of splicing and polyadenylation, we identified alternative RNA processing events specifically altered in the most aggressive alveolar form of RMS (ARMS), characterized by the upregulation of the two factors. By knockdown of YTHDC1 and DDX5, we demonstrated their role in the regulation of these RMS-specific processing events.
Extending the investigation to other tumor contexts, by analyzing data from 6679 donors, representing 19 different tumor types, we showed the correlation between the expression of the above factors and tumor-specific alternative splicing and polyadenylation events.
Altogether, our research unveils the involvement of YTHDC1 and DDX5 in the regulation of tumor-specific alternative RNA processing dynamics across various malignancies.