The ATR-WEE1 kinase module inhibits the MAC complex to regulate replication stress response

Abstract

DNA damage response is a fundamental mechanism to maintain genome stability. The ATR-WEE1 kinase module plays a central role in response to replication stress. Although the ATR-WEE1 pathway has been well studied in yeasts and animals, how ATR-WEE1 functions in plants remains unclear. Through a genetic screen for suppressors of the Arabidopsis atr mutant, we found that loss of function of PRL1, a core subunit of the evolutionarily conserved MAC complex involved in alternative splicing, suppresses the hypersensitivity of atr and wee1 to replication stress. Biochemical studies revealed that WEE1 directly interacts with and phosphorylates PRL1 at Serine 145, which promotes PRL1 ubiquitination and subsequent degradation. In line with the genetic and biochemical data, replication stress induces intron retention of cell cycle genes including CYCD1;1 and CYCD3;1, which is abolished in wee1 but restored in wee1 prl1. Remarkably, co-expressing the coding sequences of CYCD1;1 and CYCD3;1 partially restores the root length and HU response in wee1 prl1. These data suggested that the ATR-WEE1 module inhibits the MAC complex to regulate replication stress responses. Our study discovered PRL1 or the MAC complex as a key downstream regulator of the ATR-WEE1 module and revealed a novel cell cycle control mechanism.

G2/M-checkpoint activation in fasciata1 rescues an aberrant S-phase checkpoint but causes genome instability

“…Through an EMS mutagenesis suppressor screen, previous work has identified two subunits of the RNase H2 complex that, when mutated, rescue the hypersensitive response of the WEE1 KO plants to HU, likely through allowing to substitute dNTP by rNTPs into the replicating DNA (Kalhorzadeh et al, 2014;Eekhout et al, 2015), again suggesting that WEE1 activity is activated in response to a depletion of the available dNTP pool. More recently rescue of the WEE1 KO plants grown on HU has been reported for mutants in the RNA splicing factor factors PRL1 and CDC5, and the F-box like 17 (FBL17) E3-ubiquitine ligase, where the HU induced cell cycle arrest activated by WEE1 has been attributed to incorrect splicing of cyclin mRNA transcripts and stabilization of CDK inhibitory proteins, respectively (Pan et al, 2021;Wang et al, 2021).…”

Connections between the Cell Cycle and the DNA Damage Response in Plants

“…It was shown that WEE1-phosphorylation of PRL1 leads to its ubiquitylation and subsequent proteasome degradation, consequently leading to the inhibition of the MAC complex, which directly affects the intron splicing of cell cycle genes among which are CYCD1;1 and CYCD1;3. Thus, upon replicative stress, the intron retention on these CYCDs results in altered proteins unable to activate CDKA;1 leading to an arrest of cell cycle progression [124]. Similarly to the animal field [130], and given that among others, SOG1, ATM and ATR, are also subject to alternative splicing, these data further support the implication of alternative splicing in the plant DDR control (reviewed in [131]).…”

“…By identifying direct substrates of the WEE1 kinase, the Yan laboratory finally shed new light on the mechanistic action of the plant WEE1 kinase [123,124] to induce a specific DDR-dependent cell cycle arrest process (Figure 3). They demonstrated that, upon replicative stress, WEE1 interacts with and phosphorylates the F-box protein FBL17 inducing its ubiquitylation and further 26S proteasome degradation [123].…”

“…The second WEE1 substrate identified in response to replicative stress [124], is the PRL1 (PLEIOTROPIC REGULATORY LOCUS 1) WD40 protein, a core subunit of the Arabidopsis MAC (MOS4-associated Complex) complex, implicated in the control of RNA intron splicing and miRNA biogenesis [129]. It was shown that WEE1-phosphorylation of PRL1 leads to its ubiquitylation and subsequent proteasome degradation, consequently leading to the inhibition of the MAC complex, which directly affects the intron splicing of cell cycle genes among which are CYCD1;1 and CYCD1;3.…”

“…2-By targeting PRL1 a subunit of the MAC complex involved in RNA splicing, WEE1 promotes its selective degradation resulting in a non-functional MAC complex. As a consequence, CYCD mRNAs are not properly spliced compromising the activation of CDKAs and the cell cycle progression [124].…”