Abstract
RNA-binding protein (RBP) TLS/FUS plays a major role in induction of phase separation/phase transition and aggregation in relation to familial amyotrophic lateral sclerosis (ALS). Recently, organelles without lipid-bilayer membrane including stress granule, Cajal body, and nucleolus are found to be formed by the phase separation. The phase separation is an event that solutions with two solvents separate into two distinctive phases. The phase separation is prone to have solid phase and forms harmful precipitation or aggregation against living cells, indicating that the phase separation has both benefit and risk on cellular programs. Thus, it is essential for utilization of the phase separation in divergent cellular programs to control or inhibit the undesirable precipitation in living cells. Here, we analyze an inhibitory mechanism of the phase separation and precipitation. Inhibition of the phase separation is one of a critical regulatory step to prevent dysregulation of the phase separation and resulting deleterious precipitations. An inhibitory agent against the phase separation, amphiphilic alcohol, 1,6-hexanediol (1,6-HD) has been reported to examine function of phase separations. Thus, affinity chromatography of biotinylated 1,6-HD is employed to identify an initial event induced by 1,6-HD. Upon successful synthesis of biotinylated compounds of 1,6-HD (bio-1,6-HD), the affinity chromatography with bio-1,6-HD has been established at this study. The bio-1,6-HD captured protein bands on SDS-PAGE gel from HeLa cell nuclear extracts. The bands were analyzed with mass spectrometric analyses, showing that the proteins should be cytoskeleton related proteins. Further analysis using specific antibodies revealed one of the bands as human beta actin. It has been shown that beta actin is involved in divergent cellular activities including the phase separation and also neuronal functions like long-term potentiation. Therefore, beta actin might initiate the 1,6-HD-induced inhibition of the precipitation, although more experiments should be required to test whether it actually works in living cells.
Highlights
RNA-binding protein TLS/FUS is a causative gene for amyotrophic lateral sclerosis (ALS) [1, 2] and frontotemporal lobar degeneration (FTLD) [3]
Addition of 1,6-HD as 10% solution to the biotinylated isoxazole (BISOX) reaction inhibited the precipitation, and at 15% of 1,6-HD, it inhibited the BISOX-induced TLS precipitation more effectively (Figure 1, lane 5 and 6). These data clearly indicate that 1,6-HD exerts inhibitory activity on the BISOX-induced phase separation and subsequent precipitation of TLS
In order to explore a regulatory mechanism of phase separation and resultant aggregation that causes neurodegenerative diseases including ALS, we have employed affinity chromatography using a phase separation inhibitor, 1,6-HD
Summary
RNA-binding protein TLS (translocated in liposarcoma)/FUS (fused in sarcoma) is a causative gene for amyotrophic lateral sclerosis (ALS) [1, 2] and frontotemporal lobar degeneration (FTLD) [3]. Clinical symptoms of ALS are characterized by the impairment of upper and lower motor neurons, indicate muscle atrophy, and gradually spreading to the muscle of whole body including respiratory system. At the late stage of the ALS, patients suffering from the difficulty of speaking and breathing terminally die from the respiratory failure [4, 5]. Around 10% of ALS is familial disease. The causative genes of familial ALS reported are the superoxide dismutase-1 [6], TDP-43 [7], and TLS [1, 2].
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