Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 59 and 39 splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level.
Transcriptional feedback loops are central to the architecture of eukaryotic circadian clocks. Models of the Arabidopsis thaliana circadian clock have emphasized transcriptional repressors, but recently, Myb-like REVEILLE (RVE) transcription factors have been established as transcriptional activators of central clock components, including PSEUDO-RESPONSE REGULATOR5 (PRR5) and TIMING OF CAB EXPRESSION1 (TOC1). We show here that NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED1 (LNK1) and LNK2, members of a small family of four LNK proteins, dynamically interact with morningexpressed oscillator components, including RVE4 and RVE8. Mutational disruption of LNK1 and LNK2 function prevents transcriptional activation of PRR5 by RVE8. The LNKs lack known DNA binding domains, yet LNK1 acts as a transcriptional activator in yeast and in planta. Chromatin immunoprecipitation shows that LNK1 is recruited to the PRR5 and TOC1 promoters in planta. We conclude that LNK1 is a transcriptional coactivator necessary for expression of the clock genes PRR5 and TOC1 through recruitment to their promoters via interaction with bona fide DNA binding proteins such as RVE4 and RVE8.
Protein ubiquitination is involved in most cellular processes. In Arabidopsis (Arabidopsis thaliana), ubiquitin-mediated protein degradation regulates the stability of key components of the circadian clock feedback loops and the photoperiodic flowering pathway. Here, we identified two ubiquitin-specific proteases, UBP12 and UBP13, involved in circadian clock and photoperiodic flowering regulation. Double mutants of ubp12 and ubp13 display pleiotropic phenotypes, including early flowering and short periodicity of circadian rhythms. In ubp12 ubp13 double mutants, CONSTANS (CO) transcript rises earlier than that of wild-type plants during the day, which leads to increased expression of FLOWERING LOCUS T. This, and analysis of ubp12 co mutants, indicates that UBP12 and UBP13 regulate photoperiodic flowering through a CO-dependent pathway. In addition, UBP12 and UBP13 regulate the circadian rhythm of clock genes, including LATE ELONGATED HYPOCOTYL, CIRCADIAN CLOCK ASSOCIATED1, and TIMING OF CAB EXPRESSION1. Furthermore, UBP12 and UBP13 are circadian controlled. Therefore, our work reveals a role for two deubiquitinases, UBP12 and UBP13, in the control of the circadian clock and photoperiodic flowering, which extends our understanding of ubiquitin in daylength measurement in higher plants.
Bioluminescence resonance energy transfer (BRET) betweenRenilla luciferase and yellow fluorescent protein has been adapted to serve as a real-time reporter on protein-protein interactions in live plant cells by using the Arabidopsis Constitutive photomorphogenesis 1 (COP1) protein as a model system. COP1 is a repressor of light signal transduction that functions as part of a nuclear E3 ubiquitin ligase. COP1 possesses a leucine-rich nuclear-exclusion signal that resides in a domain implicated in COP1 dimerization. BRET was applied in conjunction with site-directed mutagenesis to explore the respective contributions of the nuclear-exclusion and dimerization motifs to the regulation of COP1 activity in vivo. One specific mutant protein, COP1 L105A , showed increased nuclear accumulation but retained the ability to dimerize, as monitored by BRET, whereas other mutations inhibited both nuclear exclusion and COP1 dimerization. Mutant rescue and overexpression experiments indicated that nuclear exclusion of COP1 protein is a rate-limiting step in light signal transduction.dimerization ͉ photomorphogenesis ͉ nuclear export T he etiolation response of Arabidopsis seedlings germinating in darkness is mediated in part by the Constitutive photomorphogenesis 1 (COP1) protein, a repressor of light-regulated development. COP1 functions in the nucleus (1) by targeting light-regulatory transcription factors for ubiquitin-mediated degradation by the proteasome (2-5). COP1 possesses E3 ubiquitin ligase activity, presumably in conjunction with the COP1-interacting proteins CIP8 and SPA1 (4-7). Accordingly, cop1 mutant plants show transcriptional misregulation of numerous light-inducible genes in darkness (8).A single bipartite nuclear localization signal is responsible for COP1 nuclear localization and function (1, 9). Within the nucleus, COP1 localizes to subnuclear speckles (10), which also may contain ubiquitination targets and regulators of COP1 activity (4,11,12). However, COP1 is subject also to exclusion from the nucleus, mediated by a 110-residue domain referred to as a cytoplasmic localization signal (CLS), which overlaps a predicted ␣-helical coiled-coil (CC) region (9). The nuclear exclusion of a -glucuronidase-COP1 fusion protein is enhanced by light (13-15). When fused to -glucuronidase or GFP, COP1 in fact is predominantly cytoplasmic and accumulates in a cytoplasmic inclusion body.Testing the hypothesis that the nuclear exclusion of COP1 by the CLS does, in fact, down-regulate COP1 activity in Arabidopsis is not trivial, because the CLS overlaps the CC domain, which governs both COP1 dimerization (15) and targeting to nuclear speckles (10). Thus, three aspects of COP1 that are tightly linked to one another are nuclear localization and dimerization and their role in the function of COP1. To test the dimerization of COP1 after mutagenesis within the CLS, we decided to apply bioluminescence resonance energy transfer (BRET) (16). In a BRET experiment, two candidate interaction partners are tagged with the blue-lightem...
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