Supervisors info:
Παντελής Χατζής, Επιστημονικός συνεργάτης, Ιατρική Σχολή, ΕΚΠΑ
Μιχάλης Βερυκοκάκης, Επιστημονικός συνεργάτης, Ιατρική Σχολή, ΕΚΠΑ
Αντώνιος Χατζηγεωργίου, Επίκουρος Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Summary:
Advances in RNA sequencing and other technologies have led to the discovery of the important role that many long noncoding RNAs (lncRNAs) have in physiology and tumorigenesis. Recently, genome-wide studies have suggested that hundreds of functional micropeptides may be encoded by vertebrate lncRNAs. However, the microproteome has largely been overlooked in gene annotations, primarily because of the technical difficulties in successfully identifying functional small open reading frame (smORF)-encoded peptides (SEPs). Recent studies have identified smORFs in genes previously annotated as lncRNAs, which encode functional micropeptides with potential involvement in physiology, carcinogenesis, and disease.
Recent observations from our lab have led to the discovery of a novel smORF in the annotated lncRNA EPB41L4A-AS1 that encodes a micropeptide, which we termed EPB-SEP. Here, we employed novel experimental procedures to characterize EPB-SEP at multiple levels. Firstly, through proteomic analyses and immunofluorescence experiments, we demonstrated that EPB-SEP is a 13 kDa small protein of 107 amino acids that localizes in mitochondria. Staining of mitochondria with Mitotracker-Red dye revealed that EPB-SEP significantly disrupts mitochondrial membrane potential and, therefore, diminishes mitochondrial metabolic activity. Colony formation assays demonstrated that both EPB-41L4A-AS1 lncRNA and the produced EPB-SEP are critical in cell physiology and carcinogenesis, with high levels of expression suppressing cell clonogenicity and proliferation, while low levels of expression promote cell survival, proliferation, and tumorigenesis.
Proteomics analyses revealed that more than 10% of differentially expressed proteins upon EPB-SEP overexpression were mitochondrial, with a majority of downregulated proteins localized in the inner mitochondrial membrane. Additionally, gene ontology analysis demonstrated that those downregulated proteins were significantly enriched in fundamental mitochondrial pathways. Furthermore, through immunoprecipitation experiments, we reported a list of 13 putative EPB-SEP protein interactors, including the mitochondrial proteins HAX1, CLPB, and SCO2. We propose that EPB-SEP is a novel micropeptide involved in mitochondrial metabolism, cell physiology, and carcinogenesis. Further experiments will illuminate its exact functions and potential for diagnostic and therapeutic applications.
Keywords:
Micropeptides, smORF, lncRNA, Mitochondria, Carcinogenesis
