Klap, K. | Search bioRxiv

bioRxiv2021

The dual role of the RETINOBLASTOMA-RELATED protein in the DNA damage response is spatio-temporally coordinated by the interaction with LXCXE-containing proteins.

Living organisms face threats to genome integrity caused by environmental challenges or metabolic errors in proliferating cells. To avoid the spread of mutations, cell division is temporarily arrested while repair mechanisms deal with DNA lesions. Afterwards, cells either resume division or respond to unsuccessful repair by withdrawing from the cell cycle and undergoing cell differentiation or cell death. How the success rate of DNA repair connects to the execution of cell death remains incompletely known, particularly in plants. Here we provide evidence that the Arabidopsis thaliana RETINOBLASTOMA-RELATED1 (RBR) protein, shown to play structural and transcriptional functions in the DNA damage response (DDR), coordinates these processes in time by successive interactions through its B-pocket sub-domain. Upon DNA damage induction, RBR forms nuclear foci; but the N849F substitution in the B-pocket, which specifically disrupts binding to LXCXE motif-containing proteins, abolishes RBR focus formation and leads to growth arrest. After RBR focus formation, the stress-responsive gene NAC044 arrests cell division. As RBR is released from nuclear foci, it can be bound by the conserved LXCXE motif in NAC044. RBR-mediated cell survival is inhibited by the interaction with NAC044. Disruption of NAC044-RBR interaction impairs the cell death response but is less important for NAC044 mediated growth arrest. Noteworthy, unlike many RBR interactors, NAC044 binds to RBR independent of RBR phosphorylation. Our findings indicate that the availability of the RBR B-pocket to interact with LXCXE-containing proteins couples RBR DNA repair functions and RBR transcriptional functions of in the cell death program.

plant biology↗

bioRxiv2021

A phosphorylation code regulates the multi-functional protein RETINOBLASTOMA-RELATED1 in Arabidopsis thaliana

The RETINOBLASTOMA-RELATED (RBR) proteins play a central role coordinating cell division, cell differentiation and cell survival within an environmental and developmental context. These roles reflect RBR ability to engage in multiple protein-protein interactions (PPIs), which are regulated by multi-site phosphorylation. However the functional outcomes of RBR phosphorylation in multicellular organisms remain largely unexplored. Here we test the hypothesis that phosphorylation allows diversification of RBR functions in multicellular context. Using a representative collection of transgenic loss- and gain of function point mutations in RBR phosphosites, we analysed their complementation capacity in Arabidopsis thaliana root meristems. While the number of mutated residues often correlated to the phenotypic strength of RBR phosphovariants, phospho-sites contributed differentially to distinct phenotypes. For example, the pocket-domain has a greater influence on meristematic cell proliferation, whereas the C-terminal region associates to stem cell maintenance. We found combinatorial effects between the T406 phopspho-site with others in different protein domains. Moreover, a phospho-mimetic and a phospho-defective variant, both promoting cell death, indicate that RBR controls similar cell fate choices by distinct mechanisms. Thus, additivity and specificity of RBR phospho-sites fine tune RBR activity across its multiple roles. Interestingly, a mutation disrupting RBR interactions with the LXCXE motif suppresses dominant phospho-defective RBR phenotypes. By probing protein-protein interactions of RBR variants, we found that LXCXE-containing members of the DREAM complex constitute an important component of phosphorylation-regulated RBR function, but also that RBR participates in stress or environmental responses independently of its phosphorylation state. We conclude that developmental-related, but not stress- or environmental-related functions of RBR are defined and separable by a combinatorial phosphorylation code.

plant biology↗