Therefore, we were curious to ask how these RNA granules disassemble in the cytoplasm when the stress is removed. We used Saccharomyces cerevisiae as a model system as it is a relatively simple model organism that is easy to work with and can be genetically manipulated in the laboratory. Notably, numerous scientific breakthroughs on several biological processes that were carried out in yeast are found to be true in humans.
We started with a genetic screen where we individually deleted the proteins containing the RGG-repeats in the cell. We noted that the cells with deletion of Sbp1 (Single-strand nucleic acid-binding protein 1) are defective in P-body disassembly. Surprisingly, the RGG-repeats of Sbp1 are found to be essential for the disassembly of P-bodies. Edc3 (Enhancer of decapping protein 3) is a conserved P-body resident protein and has a role in mRNA degradation. We found that Sbp1 can directly bind to Edc3 with the help of its RGG repeats, as the deletion of the IDR abolishes the Edc3-Sbp1 interaction.
Edc3 protein contains a YjeF-N domain known to self-interact (i.e., it can bind to the YjeF-N domain of another Edc3 protein), which is essential for efficient P-body assembly. Our investigation highlighted that the binding of Sbp1 to Edc3 disrupts the Edc3-Edc3 self-interaction, resulting in P-body disassembly.
RNA granules represent multicomponent viscous liquid droplets that form spontaneously by liquid-liquid phase separation (LLPS). Phase-separation can be explained by mixing oil into the water, resulting in the formation of tiny oil droplet-like structures (a process called demixing), which with time fuse to become larger oil droplets in water. Phase-separation of proteins in the cytoplasm follows a similar process of demixing. It has been found that in vitro formation of phase-separated granules can be driven by proteins containing intrinsically disordered regions (IDRs). Therefore, we developed an assay to study the assembly and disassembly of the Edc3 RNA granule in vitro. We found that Edc3 can self-interact and form tiny phase-separated assemblies when added to the phase separation buffer, which grows in size and intensity in the presence of RNA and NADH. When we added Sbp1 in the phase-separated Edc3 assemblies, we found a significant decrease in size and intensity of the assembles, which did not happen when we deleted the IDR of Sbp1, suggesting a direct link between Sbp1 mediated disassembly of Edc3 RNA granule.
It has been implicated that misregulation in the cytoplasmic granule components leads to aggregates. Aberrant granule formation is implicated in various neurodegeneration, such as amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), etc. From our research, we found that low complexity sequences have the potential to disassemble cytoplasmic aggregates of EWSR1, one of the proteins responsible for the progression of neurodegeneration, and can reduce toxicity which is imparted by the persistent aberrant granule in the cell cytoplasm. Our research paved the way for a new focus on specific amino acid sequences, which might be used as a therapeutic intervention against neurodegeneration.
