Retrotransposon LINE1 remains the only active autonomous retrotransposon in the human genome, where its copies represent as much as 17% of total DNA. LINE1 activity is the cause of over 120 monogenic diseases and it is also increased in some complex diseases, e.g. in cancer. Despite this, the process of retrotransposition has not yet been fully elucidated, and the functions of the proteins encoded within the LINE1 sequence are also poorly understood. Particularly unknown are the roles of the RNA-binding protein ORF1p, which is typically located in cytoplasmic granules and colocalizes with LINE1 mRNA. In the master's thesis, we focused on the protein composition of the ORF1p granules. They were studied by immunocytochemistry in the 2102Ep cell line, in which the protein is expressed endogenously, and in several different cell lines after transfection. We checked whether they colocalize with marker proteins of P bodies or stress granules in normal conditions or upon stress. Granules of endogenous ORF1p in 2102Ep cells under normal conditions did not colocalize with any of the marker proteins, indicating that the expressions of ORF1p probably does not cause chronic stress. In contrast, in a certain proportion of ORF1p transiently transfected cells, ORF1p colocalized with P bodies and, in some cases, with stress granules. This may occur due to the protein’s overexpression, but it could as well indicate the physiological dynamics of ORF1p granules. We unequivocally confirmed that various acute exogenous stressors (NaAsO2, DTT, NaCl) cause the mobilization of endogenous as well as transiently expressed ORF1p into stress granules. Additionally, we aimed to characterize the protein composition of ORF1p granules in vivo by proteomic methods BAR and BioID, which are based on labelling of the proteins in close proximity to the target with biotin. We aimed to compare the composition of granules in unstressed and stressed cells as well as the interactomes of functional ORF1p and its inactive mutated version JM111. To implement BioID, we prepared several fusion proteins of ORF1p or JM111 with biotin ligase BioID2 or TurboID and expressed them in mammalian cells. We analysed their activity and cellular localization and, based on the obtained results, selected the most suitable constructs for further work. Biotinylated ORF1p interaction partners were successfully isolated from cell lysates by the pull-down assay. We prepared samples and controls in which ORF1p interaction partners will be identified by liquid chromatography–mass spectrometry. The results will serve as a basis for further study of the ORF1p functions, while they will also contribute to a better understanding of the complex process of retrotransposition.
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