Ricerc@Sapienza

The RNAi machinery has many functions in the eukaryotic cell, and aspects of the RNAi molecular mechanism are highly conserved between yeast and humans. The primary role ascribed to the RNAi machinery is to promote mRNA degradation within the cytoplasm in a microRNA-dependent manner. However, both Dicer and the Argonaute protein family have expanded roles in gene regulation including their relocalization to the nucleus where they participate in transcription, alternative splicing and even DNA repair. Recently, we observed a redistribution of Ago2 and Dicer to the nucleus of human dermal and WT38 fibroblasts committed to senescence by both Western-blot analysis and indirect immunofluorescence microscopy. Interestingly, co-immunofluorescence experiments on senescent cells permitted us to co-localize both Ago2 and Dicer with fibrillarin, a well-established nucleolar marker, suggesting their localization to this nuclear region. Several works show a connection between the RNAi pathway and nucleolar regulation. Indeed, both DICER and Ago2 have been shown to reside within the nucleolus and bind to copies of the rRNA gene or directly to the rRNA.

Although the RNAi machinery has been identified in the nucleus of senescence human cells , nothing is known regarding the function and the biological meaning of the RISC components in the nucleolus during the senescence of the cells. Specifically, based on both our preliminary results and the existence of studies showing both the involvement of components of the RISC pathway in the processing of the 45S rRNA precursor and the existence of RNA fragments derived from rRNA (not all of them generated from random degradation of rRNA) the main goal of this project is that to investigate whether Ago2 and Dicer have specific and/or pleiotropic functions, ranging from ribosome biogenesis to sensor of nucleolar stress, in the nucleolus of senescent cells during senescence.