Activity and regulation of vertebrate LINE-1 retrotransposons

Transposable elements (TEs) are repetitive DNA sequences that can mobilise in our genome by a process called transposition. Based on their mechanism of transposition, TEs can be subdivided into transposons and retrotransposons, which use DNA and RNA intermediates respectively. They are found in all branches of life and have been highly successful at colonising genomes along evolution, constituting up to 50% of the human genome. Although their profound influence on evolution is unquestionable, their function and influence within the organism’s biology is still largely unknown. In humans, only retrotransposons are currently active, of which exclusively the Long INterspersed Element 1 (LINE-1 or L1) can mobilise autonomously. Although there are many cellular mechanisms in place to control LINE-1, their activity has been observed during embryogenesis, in neuronal cells, and in pathological conditions (i.e. cancer). In several cases, their insertional mutagenesis has been identified as the direct cause of genetic disorders and cancers, as well as a major contributor to disease progression by driving genetic instability. Notably, a growing body of evidence suggests that L1 encoded proteins and L1 retrotransposition intermediates can also influence cellular functions, such as inducing inflammation and senescence. Understanding the mechanisms underlying L1 regulation can help us prevent their deleterious effects and improve the prognosis of patients suffering from disorders where their activity is deregulated. Our lab has recently identified Ribonuclease H2 (RNase H2), a protein frequently mutated in Aicardi-Goutières Syndrome (AGS), as a positive regulator of L1 retrotransposition. It was proposed that the interaction of both RNase H2 and L1-encoded ORF2p protein with Proliferating cell nuclear antigen (PCNA , a DNA clamp essential for DNA replication) through their PIP (PCNA Interaction Protein) motifs, lies at the basis of their mutual coordination and allows RNase H2 to fulfil its function in L1 retrotransposition. However, further research is needed to prove this model. Also, whether changes in LINE-1 retrotransposition levels contribute to the symptomology of AGS and other disorders where L1s are found to be deregulated remains unconfirmed. An easy to manipulate animal model, such as zebrafish, would be instrumental for research into these types of questions. Zebrafish are genetically very accessible and permit many research technologies unavailable for murine model. Despite the presence of polymorphic L1 insertions suggesting recent mobilization, to date no active L1 copy has been characterized in the zebrafish genome. This thesis has two distinct aims. Firstly, to elucidate the role of the PIP domain in RNase H2 and L1-ORF2p during retrotransposition. My research into this question revealed that the PIP motif of RNase H2 does not mediate the processes that underlie its function in human LINE-1 retrotransposition. Additionally, I found that very low levels of RNase H2 (<15% of WT) are sufficient to support WT levels of LINE-1 retrotransposition. The second aim was to identify active LINE-1 copies in the zebrafish genome, in order to validate this animal model for future in vivo LINE-1 research. The specific copies from the different LINE-1 families investigated in this study did not show measurable levels of retrotransposition in our experimental setting. Nonetheless, this work cannot exclude zebrafish as a potential tool for research into the biology of LINE-1 and other TEs. Additionally, a potentially valuable LINE-1 reporter system was designed for this work, allowing the assessment of translation and retrotransposition separately. In conclusion, this work contributes to our understanding of LINE-1 biology, and can be used as a stepping stone for further research into the role of RNase H2 and PCNA in LINE-1 retrotransposition, as well the exploration into whether zebrafish could be used as an animal model for TE research