Function of PIWIL3 in stem cells

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2021In humans, genome integrity is continuously threatened by exogenous and endogenous agents. Encoded within our own genetic material we express transposable elements, a major contributor to DNA instability. These elements are nucleic acid sequences that are able to move from chromosomal locationto another, a process known as transposition. Within the family of transposable elements, retrotransposons are the only acknowledged active members in humans. Their transposition is recognised to be shaping our genome throughout evolution by contributing to genetic polymorphism. However, in the majority of the cases it introduces mutations with detrimental effects that can result in diseases. Nearly 35% of human DNA mass is composed by retrotransposons and therefore, cells have evolved many regulatory mechanisms to defend the hazards of unfettered retrotransposition. This is particularly important in germ cells where mutations can be carried through to the next generation. Usually retrotransposons are epigenetically methylated, but in germ cells where the epigenetic reprogramming events take place, the silencing marks are erased and subsequently an increase in transposition rate is detected. During this process, cells have indeed to protect their genome and one of the most notorious mechanism to regulate transposable elements are a group of proteins named Pelement induced wimpy testis (PIWIs), a subfamily of Argonaute proteins which are key players in gene silencing. The members of this family have three main motifs that are responsible to associate with small non-coding RNAs, which are responsible to conduct the complex to target mRNAs by base pairing, and to disrupt these target sequences with their catalytic domain. In flies and rodents, these proteins are mostly restricted to the germline, where they interact directly with PIWI-interacting RNAs (piRNAs). piRNAs are short sequences derived from piRNA clusters. These clusters are composed by several inactive and/or defective fragments of transposable elements, which means that piRNAs originated from theses clusters are mapped to target their respective transposon. In germ cells, PIWI/piRNA complex is primarily associated to inhibit transposition. Besides that, these elements are also key players in germ cell maintenance and differentiation. We humans and other mammals, express four copies of PIWI proteins – PIWIL1-4. Interestingly, organisms from the Muridae family do not express an ortholog for PIWIL3 which delayed our understanding of its roles and importance in mammals, more specifically in humans. All PIWI proteins have been associated with cancer. However whether they act as an oncogene or as tumour suppressor depends on the nature of the tumour. A recent report demonstrated that PIWIL3 is present in bovine oocytes and early stage embryos in a complex with Tudor and KH domain-containing (TDRKH) protein docked on the mitochondria. TDRKH is a Tudor protein that is often characterized by its physical interaction with other proteins. In female germ cells of bovine, PIWIL3/TDRKH complex formation is crucial for piRNA maturation. Half of the piRNA sequences associated with the complex were described to target retrotransposons. The roles of the other half remain unknown. Since the functions of PIWI proteins are conserved throughout evolution, we expect that PIWIL3 follows the same functional profile in the human teratocarcinoma germ NT2 cell line as in bovine oocytes and early stage embryos. Additionally, it was found that PIWI proteins are not restricted to the germline, as they are also expressed in somatic cells. In particular, this protein family was identified in neurons of flies, rodents and nematodes where they play key roles in regeneration, regulation of long-term memory, neuronal development, etc. Here we demonstrate that PIWIL3 is expressed in NT2 cells, a human testicular teratocarcinoma cell line. Moreover, TDRKH is also present and localizes on the mitochondria. However, using immunocytochemistry and co-immunoprecipitation techniques TDRKH co-localization and interaction with PIWIL3 was shown to be non-existent, inconsistent to what is seen in bovine oocytes. This result implies that PIWIL3 is not associated with piRNA processing pathway. Further examination through PIWIL3 knockdown revealed a role for this protein in supressing the activity of retrotransposons, including short and long interspaced repeat elements and human endogenous retrovirus, in NT2 cells. Lastly, PIWIL3 expression does not seem to be necessary for the differentiation process of NT2 into neuronal cells

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