RNA around the clock – regulation at the RNA level in biological timing

The circadian timing system in plants synchronizes their physiological functions with the environment. This is brought about by a global control of the gene expression program with a considerable part of the transcriptome undergoing 24-h oscillations in steady-state abundance. These circadian oscillations are driven by a set of core clock proteins that generate their own 24-h rhythm through periodic feedback on their own transcription. Here we provide an update review on molecular events at the RNA level that contribute to the 24-h rhythm of the core clock proteins and shape the circadian transcriptome. We focus on RNA-based regulation in the circadian system of the model plant Arabidopsis thaliana but also discuss selected regulatory principles in other organisms

Paradigmenwechsel im Verständnis der inneren Uhr

Staiger D. Paradigmenwechsel im Verständnis der inneren Uhr. Biologie in unserer Zeit. 2005;35(2):76-77.Der 24-Stunden-Takt, der uns aufgrund der Erdrotation aufgezwungen wird, spiegelt sich in korpereigenen Rhythmen wie dem regelmaßigen Wechsel von Schlafen und Wachsein oder der tagesperiodischen Veranderung der Korpertemperatur, des Blutdrucks und der Ausschuttung von Hormonen wider. Diese Tagesrhythmen werden nicht direkt durch die Umwelt gesteuert: Wir besitzen ein endogenes Zeitmesssystem, das einen Rhythmus von etwa 24 Stunden erzeugen und aufrechterhalten kann. Im Gegensatz zu fruheren Annahmen scheint es zur Steuerung dieser inneren Rhythmen nicht einen zentralen Schrittmacher zu geben, sondern vielmehr einen Dirigenten, der ein Orchester aus individuellen Uhren in verschiedenen Korperteilen im Takt halt

The Atger3 promoter confers circadian clock-regulated transcription with peak expression at the beginning of the night

In Arabidopsis thaliana, steady-state abundance of the Atger3 transcript encoding a germin-like cell wall protein follows a circadian rhythm, reaching its highest level at the beginning of the night. As a first step towards dissecting the molecular mechanisms underlying these transcript oscillations, the Atger3 genomic locus was characterised. Transcriptional fusions of 1.8kb and 967bp Atger3 promoter fragments to the β-glucuronidase (GUS) reporter gene mediate high-amplitude circadian oscillations of the GUS transcript in transgenic Arabidopsis. 5′ deletion to −490 greatly reduces overall transcript abundance while retaining a basal oscillation. Further deletion to −299 abolishes preferential GUS expression in the evening. Taken together, these data indicate that clock-response elements contributing to high-amplitude Atger3 oscillations largely reside between −299 and −967. Histochemical staining for GUS activity indicates that the Atger3 promoter is active in cotyledons, young leaves, petioles, the inflorescence axis, pedicels, sepals, ovary, style and siliques but not in roots, petals and anther

Molecular evolution of RRM-containing proteins and glycine-rich RNA-binding proteins in plants

*Abstract*

*Background:*
In angiosperms, RNA-binding proteins with an RNA recognition motif (RRM)-type RNA interaction domain play an important role in developmental and environmental responses. Despite their pivotal role, a comprehensive analysis of their number and diversity has only been performed in _Arabidopsis_ so far.

*Results:*
Here we present a detailed phylogenetic analysis of RRM-containing proteins in plants, the red algae _Cyanidioschyzon merolae_ and cyanobacteria. We identified two major events during the diversification of the RRM in plants, one at the emergence of green plants, and the other at the water-to-land transition. We focused on proteins that combine a single RRM with a glycine-rich stretch, known as glycine-rich RNA-binding proteins (GRPs). We found that GRPs are present in cyanobacteria, however plant and cyanobacterial GRPs are not of monophyletic origin. We provide evidence that plant GRPs form a polyphyletic group.
 
*Conclusion:*
Our work provides insights into the origin of GRPs in plants. We determined that the RRM from plants and cyanobacteria do not have a common origin. We could also determine that the acquisition of the glycine-rich stretch has happened at least on three separate occasions during the evolution of GRPs. One event led to the emergence of cyanobacterial GRPs, while later acquisition events led to the emergence of GRPs in the green lineage. No GRPs were found in red or marine green algae. We found a subgroup of GRPs exclusive to land plants, and its appearance may be linked to challenges related to the water-to-land transition.
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a simple tool for the analysis of periodic cotyledon and leaf movement in Arabidopsis thaliana

Background The analysis of circadian leaf movement rhythms is a simple yet effective method to study effects of treatments or gene mutations on the circadian clock of plants. Currently, leaf movements are analysed using time lapse photography and subsequent bioinformatics analyses of leaf movements. Programs that are used for this purpose either are able to perform one function (i.e. leaf tip detection or rhythm analysis) or their function is limited to specific computational environments. We developed a leaf movement analysis tool—PALMA—that works in command line and combines image extraction with rhythm analysis using Fast Fourier transformation and non-linear least squares fitting. Results We validated PALMA in both simulated time series and in experiments using the known short period mutant sensitivity to red light reduced 1 (srr1-1). We compared PALMA with two established leaf movement analysis tools and found it to perform equally well. Finally, we tested the effect of reduced iron conditions on the leaf movement rhythms of wild type plants. Here, we found that PALMA successfully detected period lengthening under reduced iron conditions. Conclusions PALMA correctly estimated the period of both simulated and real-life leaf movement experiments. As a platform-independent console-program that unites both functions needed for the analysis of circadian leaf movements it is a valid alternative to existing leaf movement analysis tools

Global transcript profiling of transgenic plants constitutively overexpressing the RNA-binding protein AtGRP7

Streitner C, Hennig L, Korneli C, Staiger D. Global transcript profiling of transgenic plants constitutively overexpressing the RNA-binding protein AtGRP7. BMC Plant Biology. 2010;10(1): 221.Background: The clock-controlled RNA-binding protein AtGRP7 influences circadian oscillations of its own transcript at the post-transcriptional level. To identify additional targets that are regulated by AtGRP7, transcript profiles of transgenic plants constitutively overexpressing AtGRP7 (AtGRP7-ox) and wild type plants were compared. Results: Approximately 1.4% of the transcripts represented on the Affymetrix ATH1 microarray showed changes in steady-state abundance upon AtGRP7 overexpression. One third of the differentially expressed genes are controlled by the circadian clock, and they show a distinct bias of their phase: The up-regulated genes preferentially peak around dawn, roughly opposite to the AtGRP7 peak abundance whereas the down-regulated genes preferentially peak at the end of the day. Further, transcripts responsive to abiotic and biotic stimuli were enriched among AtGRP7 targets. Transcripts encoding the pathogenesis-related PR1 and PR2 proteins were elevated in AtGRP7-ox plants but not in plants overexpressing AtGRP7 with a point mutation in the RNA-binding domain, indicating that the regulation involves RNA binding activity of AtGRP7. Gene set enrichment analysis uncovered components involved in ribosome function and RNA metabolism among groups of genes upregulated in AtGRP7-ox plants, consistent with its role in post-transcriptional regulation. Conclusion: Apart from regulating a suite of circadian transcripts in a time-of-day dependent manner AtGRP7, both directly and indirectly, affects other transcripts including transcripts responsive to abiotic and biotic stimuli. This suggests a regulatory role of AtGRP7 in the output of the endogenous clock and a complex network of transcripts responsive to external stimuli downstream of the AtGRP7 autoregulatory circuit

Timing is everything - Die Innere Uhr der Pflanzen

Staiger D, Köster T. Timing is everything - Die Innere Uhr der Pflanzen. Labor & More. 2009;5:40-41

Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

Meyer K, Köster T, Staiger D. Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process. Biomolecules. 2015;5(3):1717-1740.Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance

Bioinformatic evaluation of L-arginine catabolic pathways in 24 cyanobacteria and transcriptional analysis of genes encoding enzymes of L-arginine catabolism in the cyanobacterium Synechocystis sp. PCC 6803

Schriek S, Rückert C, Staiger D, Pistorius EK, Michel K-P. Bioinformatic evaluation of L-arginine catabolic pathways in 24 cyanobacteria and transcriptional analysis of genes encoding enzymes of L-arginine catabolism in the cyanobacterium Synechocystis sp. PCC 6803. BMC Genomics. 2007;8(1): 437.BACKGROUND:So far very limited knowledge exists on L-arginine catabolism in cyanobacteria, although six major L-arginine-degrading pathways have been described for prokaryotes. Thus, we have performed a bioinformatic analysis of possible L-arginine-degrading pathways in cyanobacteria. Further, we chose Synechocystis sp. PCC 6803 for a more detailed bioinformatic analysis and for validation of the bioinformatic predictions on L-arginine catabolism with a transcript analysis.RESULTS:We have evaluated 24 cyanobacterial genomes of freshwater or marine strains for the presence of putative L-arginine-degrading enzymes. We identified an L-arginine decarboxylase pathway in all 24 strains. In addition, cyanobacteria have one or two further pathways representing either an arginase pathway or L-arginine deiminase pathway or an L-arginine oxidase/dehydrogenase pathway. An L-arginine amidinotransferase pathway as a major L-arginine-degrading pathway is not likely but can not be entirely excluded. A rather unusual finding was that the cyanobacterial L-arginine deiminases are substantially larger than the enzymes in non-photosynthetic bacteria and that they are membrane-bound. A more detailed bioinformatic analysis of Synechocystis sp. PCC 6803 revealed that three different L-arginine-degrading pathways may in principle be functional in this cyanobacterium. These are (i) an L-arginine decarboxylase pathway, (ii) an L-arginine deiminase pathway, and (iii) an L-arginine oxidase/dehydrogenase pathway. A transcript analysis of cells grown either with nitrate or L-arginine as sole N-source and with an illumination of 50 mumol photons m-2 s-1 showed that the transcripts for the first enzyme(s) of all three pathways were present, but that the transcript levels for the L-arginine deiminase and the L-arginine oxidase/dehydrogenase were substantially higher than that of the three isoenzymes of L-arginine decarboxylase.CONCLUSION:The evaluation of 24 cyanobacterial genomes revealed that five different L-arginine-degrading pathways are present in the investigated cyanobacterial species. In Synechocystis sp. PCC 6803 an L-arginine deiminase pathway and an L-arginine oxidase/dehydrogenase pathway represent the major pathways, while the L-arginine decarboxylase pathway most likely only functions in polyamine biosynthesis. The transcripts encoding the enzymes of the two major pathways were constitutively expressed with the exception of the transcript for the carbamate kinase, which was substantially up-regulated in cells grown with L-arginine

Detection of an L-amino acid dehydrogenase activity in Synechocystis sp. PCC 6803

The protein Slr0782 from Synechocystis sp. PCC 6803, which has similarity to L-amino acid oxidase from Synechococcus elongatus PCC 6301 and PCC 7942, has been characterized in part. Immunoblot blot analysis showed that Slr0782 is mainly thylakoid membrane-associated. Moreover, expression of slr0782 mRNA and Slr0782 protein were analyzed and an activity assay was developed. Utilizing toluene-permeabilized cells, an L-arginine-stimulated O2 uptake became detectable in Synechocystis sp. PCC 6803. Besides oxidizing the basic L-amino acids L-arginine, L-lysine, L-ornithine, and L-histidine, a number of other L-amino acids were also substrates, while D-amino acids were not. The best substrate was L-cysteine, and the second best was L-arginine. The L-arginine-stimulated O2 uptake was inhibited by cations. The inhibition by o-phenanthroline and salicylhydroxamic acid suggested the presence of a transition metal besides FAD in the enzyme. Moreover, it is shown that inhibitors of the respiratory electron transport chain, such as KCN and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, also inhibited the L-arginine-stimulated O2 uptake, suggesting that Slr0782 functions as an L-arginine dehydrogenase, mediating electron transfer from L-arginine into the respiratory electron transport chain utilizing O2 as electron acceptor via cytochrome oxidase. The results imply that Slr0782 is an additional substrate dehydrogenase being able to interact with the electron transport chain of the thylakoid membrane