Matrin 3 (MATR3) is a DNA/RNA-binding nuclear matrix protein involved in several
cellular processes. These include early stages of DNA damage response, transcription,
mRNA stability, alternative splicing and mRNA export. The mutation S85C in
the MATR3 gene is associated with the development of a slowly progressive form of
amyotrophic lateral sclerosis (ALS). However, the role of MATR3 in the pathogenesis of
this disease is unknown. Additionally, little is known about the protein-protein
interactions of MATR3 and its mutants, even though these interactions could be
responsible for ALS development. In the master’s thesis, we aimed to identify proteins
that interact with human MATR3 and its mutant form MATR3S85C in vivo with the BioID
method, which is based on labelling the nearby proteins with biotin. To this end, we
generated cell lines stably expressing fusion protein of MATR3 or MATR3S85C with the
biotin ligase BioID2 or BioID2 without a fusion partner. We found that fusion proteins
of MATR3 and MATR3S85C with the biotin ligase BioID2 have the same cellular
localization as endogenous MATR3 and that BioID2 is enzymatically active. After
examining the BioID2 activity and cellular localization of the expressed proteins, we
successfully isolated biotinylated interaction partners of MATR3 and MATR3S85C from
cell lysates by the pull-down assay. Using silver staining and immunodetection of the
biotinylated proteins after Western blotting, we detected proteins specific for samples
with MATR3 and MATR3S85C, but not for samples with BioID2 without a fusion partner.
These interaction partners will be identified by liquid chromatography – mass
spectrometry. Our results will help to understand MATR3 and MATR3S85C interactome.
Moreover, we have already detected two known MATR3 interaction partners, TDP-43
and NONO in eluates with MATR3 and MATR3S85C. Finally, we have examined the
effect of MATR3S85C on stress granule formation, and based on the results, we have
concluded that this form of MATR3 prevents their formation.
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