Regulation of Abiotic Stress Signalling by Arabidopsis C-Terminal Domain Phosphatase-Like 1 Requires Interaction with a K-Homology Domain-Containing Protein

Abstract

Arabidopsis thaliana CARBOXYL-TERMINAL DOMAIN (CTD) PHOSPHATASE-LIKE 1 (CPL1) regulates plant transcriptional responses to diverse stress signals. Unlike typical CTD phosphatases, CPL1 contains two double-stranded (ds) RNA binding motifs (dsRBMs) at its C-terminus. Some dsRBMs can bind to dsRNA and/or other proteins, but the function of the CPL1 dsRBMs has remained obscure. Here, we report identification of REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) as a CPL1-interacting protein. RCF3 co-purified with tandem-affinity-tagged CPL1 from cultured Arabidopsis cells and contains multiple K-homology (KH) domains, which were predicted to be important for binding to single-stranded DNA/RNA. Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation analyses established that CPL1 and RCF3 strongly associate in vivo, an interaction mediated by the dsRBM1 of CPL1 and the KH3/KH4 domains of RCF3. Mapping of functional regions of CPL1 indicated that CPL1 in vivo function requires the dsRBM1, catalytic activity, and nuclear targeting of CPL1. Gene expression profiles of rcf3 and cpl1 mutants were similar during iron deficiency, but were distinct during the cold response. These results suggest that tethering CPL1 to RCF3 via dsRBM1 is part of the mechanism that confers specificity to CPL1-mediated transcriptional regulation.

Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super‐expression system for structural studies

“… All the TOF‐MS/MS data were searched against the UniProt protein sequence database using the GPS Explorer (Thermo Fisher, Waltham, MA, USA) software (Jeong et al ., ). …”

“…Also, putative OT subunits identified in the Arabidopsis genome by sequence homology, i.e. OST1b (At1g76400), OST2 (DAD1, At1g32210), OST4a (At5g02502) and OST4b (At3g12587), were included All the TOF-MS/MS data were searched against the UniProt protein sequence database using the GPS Explorer (Thermo Fisher, Waltham, MA, USA) software (Jeong et al, 2013). in the analyses. OST3/6 was excluded from this study because the interaction between STT3a and OST3/6 had already been reported (Farid et al, 2013;Muller et al, 2016).…”

“…Protein identification by MS was conducted as described previously (Jeong et al, 2013;Fukudome et al, 2014). The detailed procedure is provided in Methods S1.…”

Keep calm and carry on: miRNA biogenesis under stress

“…Besides making the protein inactive, HYL1 phosphorylation restricts the protein subcellular localization to the nucleus, protecting it from proteolytic degradation triggered during stressful periods of limited light (Cho et al ., ; Achkar et al ., ). Regulator of CBF Gene Expression 3 (RCF3, also known as HOS5 or SHINY), the expression of which is reduced by salt, hyperosmotic stress and ABA, assists CPL1 during HYL1 de‐phosphorylation in a tissue‐specific manner (Guan et al ., ; Jeong et al ., ; Jiang et al ., ; Chen et al ., ; Karlsson et al ., ). CPL1 and SnRK2s can also alter the phosphorylation status of SE and RCF3 with still unclear physiological and molecular effects (Chen et al ., , ; Wang et al ., ; Yan et al ., ).…”

Molecular mechanisms governing Arabidopsis iron uptake

“…For several of these genes, the effect is visible only in the first days of Fe deficiency [53]. CPL1 has been shown to interact with REGULATOR OF CBF GENE EXPRESSION 3 (RCF3), the absence of which also leads to an accumulation of Fe-responsive mRNAs [54] ( Figure 1B). However, reporter gene fusion experiments have shown that CPL1 is not expressed in the root epidermis where FRO2 and IRT1 are active [53].…”