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Decoys provide a scalable platform for the identification of plant E3 ubiquitin ligases that regulate circadian function.

ABSTRACT: The circadian clock relies on regulated degradation of clock proteins to maintain rhythmicity. Despite this, we know few components that mediate protein degradation. This is due to high levels of functional redundancy within plant E3 ubiquitin ligase families. In order to overcome this issue and discover E3 ubiquitin ligases that control circadian function, we generated a library of transgenic Arabidopsis plants expressing dominant-negative 'decoy' E3 ubiquitin ligases. We determined their effects on the circadian clock and identified dozens of new potential regulators of circadian function. To demonstrate the potency of the decoy screening methodology to overcome redundancy and identify bona fide clock regulators, we performed follow-up studies on MAC3A (PUB59) and MAC3B (PUB60). We show that they redundantly control circadian period by regulating splicing. This work demonstrates the viability of ubiquitin ligase decoys as a screening platform to overcome genetic challenges and discover E3 ubiquitin ligases that regulate plant development.

SUBMITTER: Feke A

PROVIDER: S-EPMC6483598 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Decoys provide a scalable platform for the identification of plant E3 ubiquitin ligases that regulate circadian function.

Feke Ann A Liu Wei W Hong Jing J Li Man-Wah MW Lee Chin-Mei CM Zhou Elton K EK Gendron Joshua M JM

eLife 20190405

The circadian clock relies on regulated degradation of clock proteins to maintain rhythmicity. Despite this, we know few components that mediate protein degradation. This is due to high levels of functional redundancy within plant E3 ubiquitin ligase families. In order to overcome this issue and discover E3 ubiquitin ligases that control circadian function, we generated a library of transgenic Arabidopsis plants expressing dominant-negative 'decoy' E3 ubiquitin ligases. We determined their effec ...[more]

PMID: 30950791

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Project description:Core histone proteins are essential for packaging the genomic DNA into chromatin in all eukaryotes. Since multiple genes encode these histone proteins, there is potential for generating more histones than what is required for chromatin assembly. The positively charged histones have a very high affinity for negatively charged molecules such as DNA, and any excess of histone proteins results in deleterious effects on genomic stability and cell viability. Hence, histone levels are known to be tightly regulated via transcriptional, posttranscriptional and posttranslational mechanisms. We have previously elucidated the posttranslational regulation of histone protein levels by the ubiquitin-proteasome pathway involving the E2 ubiquitin conjugating enzymes Ubc4/5 and the HECT (Homologous to E6-AP C-Terminus) domain containing E3 ligase Tom1 in the budding yeast. Here we report the identification of four additional E3 ligases containing the RING (Really Interesting New Gene) finger domains that are involved in the ubiquitylation and subsequent degradation of excess histones in yeast. These E3 ligases are Pep5, Snt2 as well as two previously uncharacterized Open Reading Frames (ORFs) YKR017C and YDR266C that we have named Hel1 and Hel2 (for Histone E3 Ligases) respectively. Mutants lacking these E3 ligases are sensitive to histone overexpression as they fail to degrade excess histones and accumulate high levels of endogenous histones on histone chaperones. Co-immunoprecipitation assays showed that these E3 ligases interact with the major E2 enzyme Ubc4 that is involved in the degradation related ubiquitylation of histones. Using mutagenesis we further demonstrate that the RING domains of Hel1, Hel2 and Snt2 are required for histone regulation. Lastly, mutants corresponding to Hel1, Hel2 and Pep5 are sensitive to replication inhibitors. Overall, our results highlight the importance of posttranslational histone regulatory mechanisms that employ multiple E3 ubiquitin ligases to ensure excess histone degradation and thus contribute to the maintenance of genomic stability.

Dataset Information

Decoys provide a scalable platform for the identification of plant E3 ubiquitin ligases that regulate circadian function.

Publications

Decoys provide a scalable platform for the identification of plant E3 ubiquitin ligases that regulate circadian function.

Similar Datasets

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