The role of liquid-liquid phase separation in the function of the NAC domain of the SOG1 protein from Arabidopsis thaliana

Samenvatting

Having a sessile lifestyle, plants are highly susceptible to DNA damage from external factors like toxic aluminum in acidic soils, which is a worldwide issue. Several mechanisms in the DNA damage response (DDR) pathway work to reduce DNA damage, but these processes can be unwanted in agriculture due to their negative impact on crop yields. Better understanding of the DRR in plants could assist in resolving this issue. For animals and humans, the p53 transcription factor is the central regulator of their DDR. SOG1, a NAC transcription factor, is a fully functional homologue of p53 in Arabidopsis thaliana. The C-terminal intrinsically disordered domain (IDR) and the DNA-binding domain of SOG1 makes it a good candidate for liquid-liquid phase separation (LLPS), which is the phenomenon underlying the formation of membraneless organelles (MLOs) in cells.
The main goal of this study is to discover the role of LLPS in the function of the NAC domain of SOG1. The focus lays on the characterization of in vitro LLPS with DNA and RNA. The importance of DNA-binding of this domain on LLPS is studied, particularly of the DNA-loop and RKRR regions of NAC. In addition, the influence of dimerization of the NAC domain is observed. In vivo, the aim is to find how NAC is organized in the cell and the effect upon stress induction. A second objective is to assess the role of the N-terminal domain of SOG1 in LLPS in vitro and in vivo.
The first finding is that the NAC domain of SOG1 can form droplets in vitro by use of phase separation with RNA (poly-A), which was confirmed with LLPS profiles on turbidity assay graphs and on fluorescence microscopy images. However, the phase separation range is limited by high RNA concentrations, which can inhibit LLPS by the reentrant phase transition behavior principle. RNA-induced LLPS is proven to be dependent on electrostatic interactions, as NaCl concentrations higher than 150 mM inhibit phase separation. Apart from RNA, the NAC domain can also phase separate with DNA, forming irregular structures with DNA promotors that are highly dynamic according to FRAP measurements. The DNA-binding ability of the NAC domain is showed to be directly linked to LLPS as is dimerization of NAC. Mutants hindered in DNA-binding only form occasional small droplets in presence of DNA or RNA. When the N-terminal domain (NTD-NAC) of SOG1 is coupled to the NAC domain, larger droplets are formed under the microscope. In addition, NTD-NAC has a bigger range of conditions under which it can undergo phase separation.
In vivo, NAC and NTD-NAC localize mainly in the nucleus and with even higher intensity in the nucleolus. Overexpression of the constructs causes appearance foci in the nucleus, possibly either due to LLPS or protein aggregation, but FRAP measurements show a high mobility of the proteins which could exclude aggregation. The induction of drought, UV, and zeocin stress on agroinfiltrated Nicotiana benthamiana leaves does not seem to alter the organization of NAC and NTD-NAC in cells.
The NAC domain of SOG1 is thus capable of undergoing LLPS in combination with RNA and DNA without the intrinsically disordered C-terminal region of SOG1. However, we suggest the disordered C-terminal domain to have an important role in the regulation of the LLPS mechanism of SOG1 upon DNA stress. Addition of the N-terminal domain causes a higher LLPS tendency of NAC giving this domain a role in LLPS. A type of interactions responsible for the LLPS with NAC is the electrostatic interaction between DNA/RNA and the DNA-binding regions of NAC.

Datum prijs	1 jul. 2024
Originele taal	English