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A cress plant's inner clock teaches us about our own
In December, the Nobel Prize for Medicine and Physiology will be awarded for the identification of genes that control our inner clock. The academics being honored examined fruit flies to determine the biorhythm. Biochemist Professor Dr. Dorothee Staiger of Bielefeld University, Saxony-Anhalt, Germany, has been researching the inner clock of plants for twenty years. Her team has now published their new study, an Open Access Article in the research journal Genome Biology. One finding — not only the inner clock, but also a protein acting as an "auxiliary clock" ensures that recurring routines take place in cells.
"The inner clock ensures that a plant adapts its metabolism to the environment in good time. Thus enabling it to directly use the first rays of the sun for photosynthesis to produce carbohydrates."
As the Nobel Prize winners have shown, individual genes in the genome of plants, animals and humans control our inner circadian clocks. Messenger molecules, messenger RNAs or mRNAs, are produced by these genes at specific times of the day. They iniate the formation of clock proteins, which reach their highest concentration at several times of day.
Clock proteins switch their own genes on and off at 24-hour intervals. They are therefore responsible for their own rhythm. They also ensure that other genes in the cell are active at the best possible time of day. They initiate different processes at certain times of the day: from opening the flowers and defending against pathogens in plants to the sleep-wake rhythm in humans.
Now Staiger and her team have examined another part of the inner clock in detail, using the model plant Arabidopsis thaliana (thale cress). During this process, they found an "auxiliary clock" - a protein called "AtGRP7".
"Interestingly, the AtGRP7 protein behaves almost like a clock protein - it influences its own 24-hour rhythm," expains Dr. Tino Köster. "As a result, the amount of AtGRP7 protein rises during the day and falls again at night." Köster and his colleague Katja Meyer are the lead authors of the study.
According to the researchers, a daily recurring cycle that can be divided into three phases is responsible for this. "In the first phase, the protein binds to its own messenger RNA and breaks it down at night. In the second phase, the reduction in messenger RNA causes less of the AtGRP7 protein to be formed. In the third phase, the diminished amount of protein ensures that new messenger RNA can form again. This marks the beginning of the cycle all over again," says Katja Meyer, who is doing her doctorate in Staiger's "RNA Biology and Molecular Physiology" research team. The scientific work of both Meyer and Tino Köster was funded by the German Academic Scholarship Foundation for several years.
A new finding is that the AtGRP7 protein not only binds to its own messenger RNA, but is capable of blocking a lot of other cell messenger RNAs.
For this, Staiger's team and their cooperation partners at the University Halle-Wittenberg had to find all the messenger RNAs in the cells of the plants on which the protein is located. In addition, the biologists subjected the plant to irradiation with ultraviolet light for about two minutes. This results in the messenger RNAs bonding firmly with the protein. They then isolated the protein and identified the RNAs bound to it by means of high-throughput sequencing. This new method is called iCLIP. It was originally developed for animal cell cultures. "For the new study, we were the world's first research team to apply the iCLIP method to whole plants," adds Dorothee Staiger.
In a further step, researchers examined what the protein does with the bound messenger RNAs in the cell. For the analysis, they artificially increased the amount of AtGRP7 protein in several plants and examined the effects of this on the messenger RNAs.
"We were able to show that an increased amount of AtGRP7 can disrupt the rhythm of some messenger RNAs. This means AtGRP7 acts as an auxiliary clock, mediating between the inner clock and the messenger RNAs dependent on the time of day."
Dorothee Staiger:"Our aim is to understand the basic interrelationships in nature. In this case, we learn how the inner clock ensures that further smaller clocks are set in motion. And we learn which strategies plants use to adapt to changing environmental conditions."
Functions for RNA-binding proteins in orchestrating plant development and environmental responses are well established. However, the lack of a genome-wide view of their in vivo binding targets and binding landscapes represents a gap in understanding the mode of action of plant RNA-binding proteins. Here, we adapt individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) genome-wide to determine the binding repertoire of the circadian clock-regulated Arabidopsis thaliana glycine-rich RNA-binding protein AtGRP7.
iCLIP identifies 858 transcripts with significantly enriched crosslink sites in plants expressing AtGRP7-GFP that are absent in plants expressing an RNA-binding-dead AtGRP7 variant or GFP alone. To independently validate the targets, we performed RNA immunoprecipitation (RIP)-sequencing of AtGRP7-GFP plants subjected to formaldehyde fixation. Of the iCLIP targets, 452 were also identified by RIP-seq and represent a set of high-confidence binders. AtGRP7 can bind to all transcript regions, with a preference for 3? untranslated regions. In the vicinity of crosslink sites, U/C-rich motifs are overrepresented. Cross-referencing the targets against transcriptome changes in AtGRP7 loss-of-function mutants or AtGRP7-overexpressing plants reveals a predominantly negative effect of AtGRP7 on its targets. In particular, elevated AtGRP7 levels lead to damping of circadian oscillations of transcripts, including DORMANCY/AUXIN ASSOCIATED FAMILY PROTEIN2 and CCR-LIKE. Furthermore, several targets show changes in alternative splicing or polyadenylation in response to altered AtGRP7 levels.
We have established iCLIP for plants to identify target transcripts of the RNA-binding protein AtGRP7. This paves the way to investigate the dynamics of posttranscriptional networks in response to exogenous and endogenous cues.
Authors: Katja Meyer, Tino Köster, Christine Nolte, Claus Weinholdt, Martin Lewinski, Ivo Grosse and Dorothee Staiger
Keywords: Circadian rhythm;Individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP); RNA immunoprecipitation (RIP); RNA-binding protein.
The study was funded by the German Research Foundation (DFG) and serves basic research.
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A. thaliana is a popular model organism in plant biology and genetics. It has a relatively small genome of approximately 135 megabase pairs (Mbp), and was the first plant to have its genome sequenced. It is a popular tool for understanding the molecular biology of many plant traits such as light sensing.
Image credit: Wikipedia