PRELIMINARY INSIGHTS INTO UNUSUAL PATHWAYS IN SENESCENCE: RNA LIGASE AND MITOCHONDRIAL MICROPROTEINS

Cellular senescence is a fundamental biological process that underlies organismal aging and the emergence of degenerative disease. While conventional models attribute senescence primarily to DNA damage, telomere erosion, and oxidative stress, there is now evidence pointing toward additional, nonconventional molecular pathways that sustain the senescent state. This thesis investigates two pathways, including human RNA ligase (C12ORF29) and mitochondrial-derived microproteins (MDPs) containing HUMANIN, SHLP2, and SHLP3 as novel cellular stress response and senescence regulators in mesenchymal stromal cells (MSCs). We found that C12ORF29 is significantly upregulated at both mRNA and protein levels under oxidative stress and cellular senescence, suggesting its direct involvement in maintaining RNA stability and ribosomal function under stressful conditions. Knockdown of C12ORF29 with siRNA induced massive cell death in senescent MSCs, suggesting its indispensable role in RNA stability and cellular survival during stress adaptation. Simultaneously, mitochondrial-derived microproteins were found to exhibit both mitochondrial and cytoplasmic localization. Oxidative stress triggered the strong upregulation of HUMANIN, SHLP2, and SHLP3 expression, as confirmed by immunocytochemistry, Western blotting, and RT qPCR analysis of RNR1 and RNR2, mitochondrial rRNA genes. Exogenous treatment with HUMANIN inhibited H₂O₂- induced apoptosis, increased proliferation, restored normal cell cycle progression, and reduced senescence-associated β-galactosidase activity. These findings confirm the cytoprotective and anti-senescent activities of HUMANIN in maintaining mitochondrial and cellular homeostasis. Together, these results suggest that C12ORF29 and mitochondrial microproteins act as cooperative factors within the cellular defence network, linking RNA maintenance and mitochondrial signalling to stress resistance and longevity. The study positions these molecules in the spotlight as potential biomarkers and therapeutic targets for protection from oxidative damage and senescence-associated dysfunction, offering new insight into the noncanonical molecular mechanisms of aging.