Paraspeckles are membraneless organelles scaffolded by the long non-coding RNA NEAT1 (nuclear paraspeckle assembly transcript 1). Specific RNA-binding proteins, such as fused in sarcoma (FUS), non-POU domain-containing octamer-binding protein (NONO) and splicing factor proline and glutamine rich (SFPQ), bind to this RNA scaffold. Through the process of liquid-liquid phase separation (LLPS), these proteins interact with NEAT1 molecules to form spherical condensates. Upon stress, poly(A)-binding protein cytoplasmic 1 (PABPC1), which normally acts as a building block of cytoplasmic stress granules, can also be recruited to nuclear paraspeckles. In amyotrophic lateral sclerosis (ALS), mutations in the C9orf72 gene lead to an expanded number of hexanucleotide repeats (G$_4$C$_2$)$_n$. The resulting mutant RNA molecules form pathological aggregates in the nucleus, which act as an alternative scaffold for paraspeckle proteins. This leads to their sequestration thereby reducing their availability for essential cellular processes. In this bachelor's thesis, we investigated the interactions between paraspeckle proteins (FUS, PABPC1, NONO, and SFPQ) and the RNA construct (G$_4$C$_2$)$_{48}$ at various molar ratios to gain insight into the nature of their interactions. By measuring turbidity and performing NaDS-PAGE analysis, we confirmed the effect of the (G$_4$C$_2$)$_{48}$ RNA construct on protein solubility, indicating RNA-protein interactions. The molar ratio proved to be crucial for the FUS protein, where a higher RNA concentration significantly reduced aggregation. For future research, interaction analyses should be performed with the remaining RNA-binding proteins under different experimental conditions.
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