Dottorato in BIOLOGIA CELLULARE E DELLO SVILUPPO

Titolo della tesi: Yeast decapping mutants as model systems for ageing and autophagy

The S. cerevisiae yeast has been successfully established over time as a useful eukaryotic model for the study of various physiological and pathological cellular processes. Among these, cellular ageing plays a role of great interest, as it is the result of the occurrence of different pathways and molecular changes which can be challenging to study in a more complex model system. In previous studies it has been shown that the mutant strain for the essential gene LSM4, whose protein product is involved in the degradation of messenger RNAs, shows premature markers of aged cells and shorter life span due to the uncontrolled accumulation of RNA in the cytoplasm. In this context, generally the survival mechanism of choice of the cell population turns out to be autophagy and, if this is not sufficient, the consequent triggered process is the regulated cell death. It has been demonstrated the involvement of LSM complex in autophagy regulation and in the case of the LSM4 mutant, a gene involved in the biosynthesis of phospholipids and membranes necessary for the autophagic process, NEM1, has a positive effect on the life span of the strain. Starting from this, in the present work we demonstrate that in the mutant strain the activation of autophagy due to restriction of nutrients or during chronological ageing is compromised, with an accumulation of cytoplasmic autophagosomal structures, and that the increase in viability u nder calorie restriction is probably dependent on a change in RNAs metabolism or in the activation of an alternative autophagic pathway . Furthermore, we created a reporter system comprising the truncated form of LSM4 and the GFP protein at genome level using the CRISPR /Cas9 system, which shows the phenotypic markers of premature aged cells as well.