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.
The main goal of our project will be to identify and clarify a new functional role of Ago2 and Dicer, two components of the RNAi machinery, into the nucleolus representing a dynamic structure that has roles in various processes, from ribosome biogenesis to regulation of the cell cycle and the cellular stress response. Despite their nuclear localization, only few works have identified Ago2 and Dicer as part of the nucleolar compartments (1, 2, 3). It is important to remind that RNAi is a conserved genome defense mechanism in eukaryotes that protects against deleterious effects of repetitive sequences such as transposons and viral genomes (4). Despite this assumption, very few reports have explored the relationship between RNAi machinery with nucleoli, which are composed by a set of tandem-repeated genes. To this regard, our study provides important insights into the knowledge of the molecular mechanism subtended to the peculiar repetitive ribosomal DNA locus (rDNA) maintenance.
Our multitasking approach about the Ago2 and Dicer relocation into the senescent nucleoli will contribute to shed in light the functional role of the RNAi machinery in rDNA expression, rRNA processing, ribosome biogenesis. This panorama could open new frontline about the treatment of these pathologies.
Finally, our deepen research about the role of Ago2 and Dicer in the context of senescent-stressed nucleoli may represent a pioneering work that mark the way to explore the details of the mechanistic events and protein effectors regulating cellular senescence. Biological and medical research has developed a great deal of attention to cellular senescence since the senescence program is implicated in diverse biological processes. Interestingly, it has been proposed that senescent cells generated early in life provide an advantage during development, tissue regeneration, and by inhibiting neoplastic transformation, but aberrant and chronic accumulation of senescent cells late in life drives various features of ageing, including age-related disease and tissue deterioration (5).
In the early stages of cutaneous wound healing, senescent cells induce myofibroblast differentiation and promote wound closure; during embryogenesis their transient presence participate to the correct organismal developed (6). After performing their duty in young organisms, senescent cells are able to induce attraction, and activation of immune cells that can result in senescent-cell clearance through a process called immune surveillance. Differently, it has been shown in old organisms that age-related senescent cell accumulation coincides with reduced immune system function, suggesting that impaired immune function allows senescent cells to evade clearance (7). This accumulation of senescent cells over time shortens lifespan, promotes tissue deterioration, and impairs the function of several organs providing the basis for age¿related pathologies (8).
Lastly, senescent cells have a dual role on tumor progression. In fact, if on the one hand, senescence is a potent tumour-suppressor mechanism that prevents the expansion of damaged and preneoplastic cells, on the other hand, malignant cancers exploit the secretome of senescent cells to stimulate growth, angiogenesis, epithelial-to-mesenchymal transition (EMT), immune cell evasion, and metastasis (5).
For all these reasons, a better comprehension of the genetic networks that control and induce senescence will be important not only to improve our basic knowledge into cell cycle and the cellular stress response. It may constitute the start point to develop new therapeutic approaches linked to wound healing, to developmental problems, age¿related pathologies and cancer.
Our research focused on the role of Dicer and Ago2 proteins in the nucleolar pathways linked to senescent could provide insight on new unexplored molecular mechanisms through which to counteract and/or modulate the above mentioned physiological and pathological processes.
As putative drivers of senescence, Ago2 and Dicer may be considered as promising new targets for a therapeutic intervention known as senotherapy that has potential to improve tumor outcome rather than attenuate age-related dysfunctions.
Overall this project will contribute to the current understanding of the molecular mechanisms underlying senescence, laying the foundations for a plethora of new therapeutic approaches.
1. Catalanotto C, et al. Int J Mol Sci. 2016 17(10).
2. Sinkkonen L, et al. PLoS One 2010 Aug 13;5(8):e12175.
3. Atwood BL, et al. J Biol Chem 2016 Aug 19;291(34):17919-28.
4. Buchon N and Vaury C. Heredity. 2006 96:159-202.
5. He S. et al. Cell. 2017 169 (6): 1000-1011.
6. Demaria, M. et al. Dev. Cell 2014 31:722¿733. ¿
7. Iannello, A. J. Exp. Med. 2013 210:2057¿2069.
8. Baker, D. J. et al. Nature. 2016 530:184¿189.