ALS is a fatal neurodegenerative disease characterized by gradual degeneration and eventual loss of motor neurons, which results in progressive muscular paralysis. Even though previous research discovered multiple genetic mutations that contribute to the disease, the molecular mechanisms of ALS are still largely not understood. One of the most common causal mutations in ALS is a hexanucleotide repeat expansion (HRE) GGGGCC in the gene C9ORF72. Due to the high G content, this sequence repeats can form higher order structures, including multiple types of G-quadruplexes (GQs). There are many pathways through which HRE in C9ORF72 could initiate neurodegeneration. Aberrant multivalent interactions of RNA with RNA-binding proteins (RBPs) could cause sequestration of RBPs in nuclear and cytoplasmic foci, inhibiting their physiological function. Splicing regulator hnRNP H1 was identified as one of the main interactors of G4C2 nucleotide repeats. It was also found in the nuclear granules, which have been observed in C9ORF72 ALS. The nature of hnRNP H1 binding to G4C2 repeats remains ambiguous. In my master’s thesis, we wanted to determine the structural aspects of hnRNP H1 binding to G4C2 repeats. First, we aimed to show a preference in binding to either folded or unfolded GQs. Next, we were interested if hnRNP H1 possesses the ability to linearize folded GQs. With IVA cloning, we prepared a vector construct pMCSG7-hnRNPH1-MBP, which we then used for the protein expression in bacterial cells. We then purified the protein using His-trap and size exclusion chromatography. After size exclusion chromatography, we cleaved off the fusion partner MBP with TEV protease and then performed heparin chromatography. We obtained a pure protein which we could use for the following interaction assays. To determine whether the protein binds to the linear or GQ structure we used thermophoresis. Along with this we performed fluorescence quenching assay which would allow us to observe the protein’s ability to unfold GQs. For this, we used a ssDNA oligonucleotide which contained Cy5 on 5’ end of the sequence and BHQ2 on its 3’. The results of thermophoresis and fluorescence quenching assay imply a preferential binding to linear G4C2 repeats. In these two assays, we could not detect any binding to GQs, which prevented us from determining if hnRNP H1 possesses the ability to linearize these structures. To additionally confirm the binding of hnRNP H1 to G4C2 repeats, we performed electrophoretic mobility shift assays (EMSA). For this, we used 4× G4C2 RNA repeats, which we prepared with in vitro transcription. The results of EMSA implicate a formation of higher molecular weight structure, which could form through the mechanism of liquid-liquid phase separation.
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