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Post-transcriptional regulation makes an important contribution to adjusting the transcriptome to environmental changes in plants. RNA-binding proteins are key players that interact specifically with mRNAs to co-ordinate their fate. While the regulatory interactions between proteins and RNA are well understood in animals, until recently little information was available on the global binding landscape of RNA-binding proteins in higher plants. This is not least due to technical challenges in plants. In turn, while numerous RNA-binding proteins have been identified through mutant analysis and homology-based searches in plants, only recently a full compendium of proteins with RNA-binding activity has been experimentally determined for the reference plant Arabidopsis thaliana. State-of-the-art techniques to determine RNA-protein interactions genome-wide in animals are based on the covalent fixation of RNA and protein in vivo by UV light. This has only recently been successfully applied to plants. Here, we present practical considerations on the application of UV irradiation based methods to comprehensively determine in vivo RNA-protein interactions in Arabidopsis thaliana, focussing on individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) and mRNA interactome capture. Copyright © 2019 Elsevier Inc. All rights reserved.

The timing of floral initiation is a tightly controlled process in plants. The circadian clock regulated glycine-rich RNA-binding protein AtGRP7, a known regulator of splicing, was previously shown to regulate flowering time mainly by affecting the MADS-box repressor FLC. Loss of AtGRP7 leads to elevated FLC expression and late flowering in the atgrp7-1 mutant. Here, we analyze genetic interactions of AtGRP7 with key regulators of the autonomous and the thermosensory pathway of floral induction. RNAi mediated reduction of the level of the paralogous AtGRP8 in atgrp7-1 further delays floral transition compared to atgrp7-1. AtGRP7 acts in parallel to FCA, FPA and FLK in the branch of the autonomous pathway comprised of RNA-binding proteins. It acts in the same branch as FLD, and AtGRP7 loss-of-function mutants show elevated levels of di-methylated lysine 4 of histone H3, a mark for active transcription. In addition to its role in the autonomous pathway, AtGRP7 acts in the thermosensory pathway of flowering time control by regulating alternative splicing of the floral repressor FLM. Overexpression of AtGRP7 selectively favors formation of the repressive isoform FLM-beta. Our results suggest that the RNA-binding proteins AtGRP7 and AtGRP8 influence MADS-Box transcription factors in at least two different pathways of flowering time control. This highlights the importance of RNA-binding proteins to fine-tune the integration of varying cues into flowering time control and further strengthens the view that the different pathways, although genetically separable, constitute a tightly interwoven network to ensure plant reproductive success under changing environmental conditions. © The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

In recent years, it has become increasingly recognized that regulation at the RNA level pervasively shapes the transcriptome in eukaryotic cells. This has fostered an interest in the mode of action of RNA-binding proteins that, via interaction with specific RNA sequence motifs, modulate gene expression. Understanding such posttranscriptional networks controlled by an RNA-binding protein requires a comprehensive identification of its in vivo targets. In metazoans and yeast, methods have been devised to stabilize RNA-protein interactions by UV cross-linking before isolating RNA-protein complexes using antibodies, followed by identification of associated RNAs by next-generation sequencing. These methods are collectively referred to as CLIP-Seq (cross-linking immunoprecipitation-high-throughput sequencing). Here, we present a version of the individual nucleotide resolution cross-linking and immunoprecipitation procedure that is suitable for use in the model plant Arabidopsis thaliana.

BackgroundRNA-binding proteins interact with their target RNAs at specific sites. These binding sites can be determined genome-wide through individual nucleotide resolution crosslinking immunoprecipitation (iCLIP). Subsequently, the binding sites have to be visualized. So far, no visualization tool exists that is easily accessible but also supports restricted access so that data can be shared among collaborators.ResultsHere we present SEQing, a customizable interactive dashboard to visualize crosslink sites on target genes of RNA-binding proteins that have been obtained by iCLIP. Moreover, SEQing supports RNA-seq data that can be displayed in a diffrerent window tab. This allows, e.g. crossreferencing the iCLIP data with genes differentially expressed in mutants of the RBP and thus obtain some insights into a potential functional relevance of the binding sites. Additionally, detailed information on the target genes can be incorporated in another tab.ConclusionSEQing is written in Python3 and runs on Linux. The web-based access makes iCLIP data easily accessible, even with mobile devices. SEQing is customizable in many ways and has also the option to be secured by a password. The source code is available at https://github.com/malewins/SEQing.

N6-methyladenosine (m6A) emerges as an important modification in eukaryotic mRNAs. m6A has first been reported in 1974, and its functional significance in mammalian gene regulation and importance for proper development has been well established. An arsenal of writer, eraser, and reader proteins accomplish deposition, removal, and interpretation of the m6A mark, resulting in dynamic function. This led to the concept of an epitranscriptome, the compendium of RNA species with chemical modification of the nucleobases in the cell, in analogy to the epigenome. While m6A has long been known to also exist in plant mRNAs, proteins involved in m6A metabolism have only recently been detected by mutant analysis, homology search, and mRNA interactome capture in the reference plant Arabidopsis thaliana. Dysregulation of the m6A modification causes severe developmental abnormalities of leaves and roots and altered timing of reproductive development. Furthermore, m6A modification affects viral infection. Here, we discuss recent progress in identifying m6A sites transcriptome-wide, in identifying the molecular players involved in writing, removing, and reading the mark, and in assigning functions to this RNA modification in Arabidopsis thaliana. We highlight similarities and differences to m6A modification in mammals and provide an outlook on important questions that remain to be addressed. © The Author(s) 2019. Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.

Background 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. Results 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. Conclusions 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. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1332-x) contains supplementary material, which is available to authorized users.

The Arabidopsis thaliana root epidermis is comprised of two cell types, hair and nonhair cells, which differentiate from the same precursor. Although the transcriptional programs regulating these events are well studied, post-transcriptional factors functioning in this cell fate decision are mostly unknown. Here, we globally identify RNA-protein interactions and RNA secondary structure in hair and nonhair cell nuclei. This analysis reveals distinct structural and protein binding patterns across both transcriptomes, allowing identification of differential RNA binding protein (RBP) recognition sites. Using these sequences, we identify two RBPs that regulate hair cell development.Specifically, we find that SERRATE functions in a microRNA-dependent manner to inhibit hair cellfate, while also terminating growth of root hairs mostly independent of microRNA biogenesis. In addition, we show that GLYCINE-RICH PROTEIN 8 promotes hair cell fate while alleviating phosphate starvation stress. In total, this global analysis revealspost-transcriptional regulators of plant root epidermal cell fate. Copyright 2017 Elsevier Inc. All rights reserved.

The hnRNP-like glycine-rich RNA-binding protein AtGRP7 regulates pre-mRNA splicing in Arabidopsis. Here we used small RNA-seq to show that AtGRP7 also affects the miRNA inventory. AtGRP7 overexpression caused a significant reduction in the level of 30 miRNAs and an increase for 14 miRNAs with a minimum log2 fold change of ±0.5. Overaccumulation of several pri-miRNAs including pri-miR398b, pri-miR398c, pri-miR172b, pri-miR159a and pri-miR390 at the expense of the mature miRNAs suggested that AtGRP7 affects pri-miRNA processing. Indeed, RNA immunoprecipitation revealed that AtGRP7 interacts with these pri-miRNAs in vivo. Mutation of an arginine in the RNA recognition motif abrogated in vivo binding and the effect on miRNA and pri-miRNA levels, indicating that AtGRP7 inhibits processing of these pri-miRNAs by direct binding. In contrast, pri-miRNAs of selected miRNAs that were elevated or not changed in response to high AtGRP7 levels were not bound in vivo. Reduced accumulation of miR390, an initiator of trans-acting small interfering RNA (ta-siRNA) formation, also led to lower TAS3 ta-siRNA levels and increased mRNA expression of the target AUXIN RESPONSE FACTOR4. Furthermore, AtGRP7 affected splicing of pri-miR172b and pri-miR162a. Thus, AtGRP7 is an hnRNP-like protein with a role in processing of pri-miRNAs in addition to its role in pre-mRNA splicing.

Plants overexpressing the RNA-binding protein AtGRP7 (AtGRP7-ox plants) constitutively express the PR-1 (PATHOGENESIS-RELATED-1), PR-2 and PR-5 transcripts associated with salicylic acid (SA)-mediated immunity and show enhanced resistance against Pseudomonas syringae pv. tomato (Pto) DC3000. Here, we investigated whether the function of AtGRP7 in plant immunity depends on SA. Endogenous SA was elevated fivefold in AtGRP7-ox plants. The elevated PR-1, PR-2 and PR-5 levels were eliminated upon expression of the salicylate hydroxylase nahG in AtGRP7-ox plants and elevated PR-1 levels were suppressed by sid (salicylic acid deficient) 2-1 that is impaired in SA biosynthesis. RNA immunoprecipitation showed that AtGRP7 does not bind the PR-1 transcript in vivo, whereas it binds PDF1.2. Constitutive or inducible AtGRP7 overexpression increases PR-1 promoter activity, indicating that AtGRP7 affects PR-1 transcription. In line with this, the effect of AtGRP7 on PR-1 is suppressed by npr (non-expressor of PR genes) 1. Whereas AtGRP7-ox plants restricted growth of Pto DC3000 compared with wild type (wt), sid2-1 AtGRP7-ox plants allowed more growth than AtGRP7-ox plants. Furthermore, we show an enhanced hypersensitive response triggered by avirulent Pto DC3000 (AvrRpt2) in AtGRP7-ox compared with wt. In sid2-1 AtGRP7-ox, an intermediate phenotype was observed. Thus, AtGRP7 has both SA-dependent and SA-independent effects on plant immunity.

RNA-protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture - where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

This study focused on the identification and phylogenetic analysis of glycine-rich RNA binding proteins that contain an RNA recognition motif (RRM)-type RNA binding domain in addition to a region with contiguous glycine residues in representative plant species. In higher plants, glycine-rich proteins with an RRM have met considerable interest as they are responsive to environmental cues and play a role in cold tolerance, pathogen defense, flowering time control, and circadian timekeeping. To identify such RRM containing proteins in plant genomes we developed an RRM profile based on the known glycine-rich RRM containing proteins in the reference plant Arabidopsis thaliana. The application of this remodeled RRM profile that omitted sequences from non-plant species reduced the noise when searching plant genomes for RRM proteins compared to a search performed with the known RRM_1 profile. Furthermore, we developed an island scoring function to identify regions with contiguous glycine residues, using a sliding window approach. This approach tags regions in a protein sequence with a high content of the same amino acid, and repetitive structures score higher. This definition of repetitive structures in a fixed sequence length provided a new glance for characterizing patterns which cannot be easily described as regular expressions. By combining the profile-based domain search for well-conserved regions (the RRM) with a scoring technique for regions with repetitive residues we identified groups of proteins related to the A. thaliana glycine-rich RNA binding proteins in eight plant species.

I. II. III. IV. V. VI. VII. References SUMMARY: Infection by phytopathogenic bacteria triggers massive changes in plant gene expression, which are thought to be mostly a result of transcriptional reprogramming. However, evidence is accumulating that plants additionally use post-transcriptional regulation of immune-responsive mRNAs as a strategic weapon to shape the defense-related transcriptome. Cellular RNA-binding proteins regulate RNA stability, splicing or mRNA export of immune-response transcripts. In particular, mutants defective in alternative splicing of resistance genes exhibit compromised disease resistance. Furthermore, detection of bacterial pathogens induces the differential expression of small non-coding RNAs including microRNAs that impact the host defense transcriptome. Phytopathogenic bacteria in turn have evolved effector proteins to inhibit biogenesis and/or activity of cellular microRNAs. Whereas RNA silencing has long been known as an antiviral defense response, recent findings also reveal a major role of this process in antibacterial defense. Here we review the function of RNA-binding proteins and small RNA-directed post-transcriptional regulation in antibacterial defense. We mainly focus on studies that used the model system Arabidopsis thaliana and also discuss selected examples from other plants.

Alternative splicing (AS) of pre-mRNAs is an important regulatory mechanism shaping the transcriptome. In plants, only few RNA-binding proteins are known to affect AS. Here, we show that the glycine-rich RNA-binding protein AtGRP7 influences AS in Arabidopsis thaliana. Using a high-resolution RT-PCR-based AS panel, we found significant changes in the ratios of AS isoforms for 59 of 288 analyzed AS events upon ectopic AtGRP7 expression. In particular, AtGRP7 affected the choice of alternative 5' splice sites preferentially. About half of the events are also influenced by the paralog AtGRP8, indicating that AtGRP7 and AtGRP8 share a network of downstream targets. For 10 events, the AS patterns were altered in opposite directions in plants with elevated AtGRP7 level or lacking AtGRP7. Importantly, RNA immunoprecipitation from plant extracts showed that several transcripts are bound by AtGRP7 in vivo and indeed represent direct targets. Furthermore, the effect of AtGRP7 on these AS events was abrogated by mutation of a single arginine that is required for its RNA-binding activity. This indicates that AtGRP7 impacts AS of these transcripts via direct interaction. As several of the AS events are also controlled by other splicing regulators, our data begin to provide insights into an AS network in Arabidopsis.

The Pseudomonas syringae type III effector HopU1 is a mono-ADP-ribosyltransferase that is injected into plant cells by the type III protein secretion system. Inside the plant cell it suppresses immunity by modifying RNA-binding proteins including the glycine-rich RNA-binding protein GRP7. The crystal structure of HopU1 at 2.7-Å resolution reveals two unique protruding loops, L1 and L4, not found in other mono-ADP-ribosyltransferases. Site-directed mutagenesis demonstrates that these loops are essential for substrate recognition and enzymatic activity. HopU1 ADP-ribosylates the conserved arginine 49 of GRP7, and this reduces the ability of GRP7 to bind RNA in vitro. In vivo, expression of GRP7 with Arg-49 replaced with lysine does not complement the reduced immune responses of the Arabidopsis thaliana grp7-1 mutant demonstrating the importance of this residue for GRP7 function. These data provide mechanistic details how HopU1 recognizes this novel type of substrate and highlights the role of GRP7 in plant immunity.

Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state are novel tools for monitoring intracellular protein trafficking. A codon-optimized variant of the reversibly photoswitchable fluorescent protein DRONPA was designed for the use in transgenic Arabidopsis plants. Its codon usage is also well adapted to the mammalian codon usage. The synthetic protein, DRONPA-s, shows photochemical properties and switching behavior comparable to that of the original DRONPA from Pectiniidae both in vitro and in vivo. DRONPA-s fused to the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) under control of the endogenous AtGRP7 promoter localizes to cytoplasm, nucleoplasm and nucleolus of transgenic Arabidopsis plants. To monitor the intracellular transport dynamics of AtGRP7-DRONPA-s, we set up a single-molecule sensitive confocal fluorescence microscope. Fluorescence recovery after selective photoswitching experiments revealed that AtGRP7-DRONPA-s reaches the nucleus by carrier-mediated transport. Furthermore, photoactivation experiments showed that AtGRP7-DRONPA-s is exported from the nucleus. Thus, AtGRP7 is a nucleocytoplasmic shuttling protein. Our results show that the fluorescent marker DRONPA-s is a versatile tool to track protein transport dynamics in stably transformed plants.

The Arabidopsis RNA-binding protein AtGRP8 undergoes negative autoregulation at the post-transcriptional level. An elevated AtGRP8 protein level promotes the use of a cryptic 5' splice site to generate an alternatively spliced transcript, as_AtGRP8, retaining the 5' half of the intron with a premature termination codon. In mutants defective in nonsense-mediated decay (NMD) abundance of as_AtGRP8 but not its pre-mRNA is elevated, indicating that as_AtGRP8 is a direct NMD target, thus limiting the production of functional AtGRP8 protein. In addition to its own pre-mRNA, AtGRP8 negatively regulates the AtGRP7 transcript through promoting the formation of the equivalent alternatively spliced as_AtGRP7 transcript, leading to a decrease in AtGRP7 abundance. Recombinant AtGRP8 binds to its own and the AtGRP7 pre-mRNA, suggesting that this interaction is relevant for the splicing decision in vivo. AtGRP7 itself is part of a negative autoregulatory circuit that influences circadian oscillations of its own and the AtGRP8 transcript through alternative splicing linked to NMD. Thus, we identify an interlocked feedback loop through which two RNA-binding proteins autoregulate and reciprocally crossregulate by coupling unproductive splicing to NMD. A high degree of evolutionary sequence conservation in the introns retained in as_AtGRP8 or as_AtGRP7 points to an important function of these sequences.

The circadian timing system in plants synchronizes their physiological functions with the environment. This is achieved by a global control of gene expression programs 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. Additionally, post-transcriptional events are instrumental for oscillations of core clock genes and genes in clock output. Here we provide an update 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 the circadian system of the model plant Arabidopsis thaliana but also discuss selected regulatory principles in other organisms.

Control at the posttranscriptional level emerges as an important layer of regulation in the circadian timing system. RNA-binding proteins that specifically interact with cis-regulatory motifs within pre-mRNAs are key elements of this regulation. While the ability to interact with RNA in vitro has been demonstrated for numerous Arabidopsis RNA-binding proteins, a full understanding of posttranscriptional networks controlled by an RNA-binding protein requires the identification of its immediate in vivo targets. Here we describe differential RNA immunoprecipitation in transgenic Arabidopsis thaliana plants expressing RNA-binding protein variants epitope-tagged with green fluorescent protein. To control for RNAs that nonspecifically co-purify with the RNA-binding protein, transgenic plants are generated with a mutated version of the RNA-binding protein that is not capable of binding to its target RNAs. The RNA-binding protein variants are expressed under the control of their authentic promoter and cis-regulatory motifs. Incubation of the plants with formaldehyde in vivo cross-links the proteins to their RNA targets. A whole-cell extract is then prepared and subjected to immunoprecipitation with an antibody against the GFP tag and to mock precipitation with an antibody against the unrelated red fluorescent protein. The RNAs coprecipitating with the proteins are eluted from the immunoprecipitate and identified via reverse transcription-PCR.

Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased K-d value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R-49 that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding. (C) 2014 Elsevier Inc. All rights reserved.

The RNA-binding protein Arabidopsis thaliana glycine-rich RNA-binding protein 7 (AtGRP7) regulates the steady-state abundance of numerous target transcripts in A. thaliana. Here we show that the GA1 and GA2 transcripts encoding the first enzymes of the gibberellin biosynthetic pathway are expressed at reduced levels in transgenic plants ectopically over-expressing AtGRP7 (AtGRP7-ox plants). Furthermore, the levels of the bioactive phytohormone GA(4) as well as of several intermediates of the GA biosynthetic pathway are reduced in AtGRP7-ox plants. The transgenic plants show a reduced length of the vegetative stem. The application of exogenous GA largely reverses the phenotype by increasing the number of vegetative internodes. AtGRP7-ox plants flower with fewer leaves than wt plants, suggesting that the floral promotive effect of AtGRP7 bypasses the effect of a reduced GA level in AtGRP7-ox plants. Upon GA treatment, AtGRP7-ox plants flower only slightly earlier than wild type plants. Thus, exogenous GA has only a small additional effect in reducing the number of leaves at the onset of flowering in AtGRP7-ox plants.

AnArabidopsistranscript preferentially expressed at the end of the daily light period codes for the RNA–binding proteinAtGRP7. A reverse genetic approach inArabidopsis thalianahas revealed its role in the generation of circadian rhythmicity:AtGRP7 is part of a negative feedback loop through which it influences the oscillations of its own transcript. Biochemical and genetic experiments indicate a mechanism for this autoregulatory circuit:Atgrp7 gene transcription is rhythmically activated by the circadian clock during the day. TheAtGPR7 protein accumulates with a certain delay and represses further accumulation of its transcript, presumably at the post–transcriptional level. In this respect, theAtGRP7 feedback loop differs from known circadian oscillators in the fruitflyDrosophilaand mammals based on oscillating clock proteins that repress transcription of their own genes with a 24 h rhythm. It is proposed that theAtGRP7 feedback loop may act within an output pathway from theArabidopsisclock.

The endogenous clock that drives circadian rhythms is thought to communicate temporal information within the cell via cycling downstream transcripts. A transcript encoding a glycine-rich RNA-binding protein, Atgrp7 , in Arabidopsis thaliana undergoes circadian oscillations with peak levels in the evening. The At GRP7 protein also cycles with a time delay so that Atgrp7 transcript levels decline when the At GRP7 protein accumulates to high levels. After At GRP7 protein concentration has fallen to trough levels, Atgrp7 transcript starts to reaccumulate. Overexpression of At GRP7 in transgenic Arabidopsis plants severely depresses cycling of the endogenous Atgrp7 transcript. These data establish both transcript and protein as components of a negative feedback circuit capable of generating a stable oscillation. At GRP7 overexpression also depresses the oscillation of the circadian-regulated transcript encoding the related RNA-binding protein At GRP8 but does not affect the oscillation of transcripts such as cab or catalase mRNAs. We propose that the At GRP7 autoregulatory loop represents a “slave” oscillator in Arabidopsis that receives temporal information from a central “master” oscillator, conserves the rhythmicity by negative feedback, and transduces it to the output pathway by regulating a subset of clock-controlled transcripts.

High-throughput sequencing for transcript profiling in plants has revealed that alternative splicing (AS) affects a much higher proportion of the transcriptome than was previously assumed. AS is involved in most plant processes and is particularly prevalent in plants exposed to environmental stress. The identification of mutations in predicted splicing factors and spliceosomal proteins that affect cell fate, the circadian clock, plant defense, and tolerance/sensitivity to abiotic stress all point to a fundamental role of splicing/AS in plant growth, development, and responses to external cues. Splicing factors affect the AS of multiple downstream target genes, thereby transferring signals to alter gene expression via splicing factor/AS networks. The last two to three years have seen an ever-increasing number of examples of functional AS. At a time when the identification of AS in individual genes and at a global level is exploding, this review aims to bring together such examples to illustrate the extent and importance of AS, which are not always obvious from individual publications. It also aims to ensure that plant scientists are aware that AS is likely to occur in the genes that they study and that dynamic changes in AS and its consequences need to be considered routinely.

RNA-based regulation is increasingly recognized as an important factor shaping the cellular transcriptome. RNA-binding proteins that interact with cis-regulatory motifs within pre-mRNAs determine the fate of their targets. Understanding posttranscriptional networks controlled by an RNA-binding protein requires the identification of its immediate in vivo targets. Here we describe RNA immunoprecipitation in Arabidopsis thaliana. Transgenic plants expressing an RNA-binding protein fused to green fluorescent protein are treated with formaldehyde to "trap" RNAs in complexes with their physiological protein partners. A whole-cell extract is subjected to immunoprecipitation with an antibody against the GFP tag. In parallel, a mock immunoprecipitation is performed using an unrelated antibody. Coprecipitated RNAs are eluted from the immunoprecipitate and identified via real-time PCR. Enrichment relative to immunoprecipitation from plants expressing GFP only and mock immunoprecipitation with an unrelated antibody indicates specific binding.

Summary To investigate physiological processes generated by endogenous circadian rhythms on the molecular level, we have identified clock-controlled genes In the long-day plant Sinapis aiba L. A cDNA library was differentially screened using cDNA probes representing transcripts expressed at either the middle of the light period or the middle of the dark period. Two closely related groups of transcripts, Sagrp1 and Sagrp2, controlled by a circadian rhythm have been isolated. RNA blot analysis verified that transcript levels oscillate in plants grown in light/dark cycles with maxima between ‘Zeitgober’ time (zt)8 and zt12 (8–12 h after onset of illumination) and minima around zt20. Steady-state mRNA levels continue to oscillate in plants shifted from light/dark cycles to constant light. No synchronous mRNA oscillations are found in plants grown from seed in constant light at constant temperature, suggesting that the clock has to be entrained initially. In contrast, when plants grown in constant light are exposed to rhythmic temperature shifts oscillations of steady-state Sagrp mRNA levels are induced, indicating that temperature acts as an alternative external stimulus (zeitgeber) other than light to entrain the oscillator. In situ hybridization reveals that both transcript groups are expressed predominantly in meristematic and growing tissue. Strong expression is observed in the leaf primordia of the shoot apex, the procambial strands, cambium and in all cell layers of young leaves around zt12. In contrast, little or no signal is found on tissue sections isolated at zt20. This indicates that the oscillator(s) regulating Sagrp transcript fluctuations operate(s) synchronously in different organs. For both transcript groups cDNAs were isolated corresponding to unspliced pre-mRNAs or to transcripts generated by the use of a second 5′ splice site. The cDNAs corresponding to the fully spliced transcripts contain open reading frames for polypeptides of 16 kDa, each containing a putative N-terminal RNA recognition motif and a C-terminal region rich in glycine. The predicted proteins show strong homology to an ABA-inducible glycine-rich protein from maize embryos and to the mammalian RNA-binding protein A1 of the heterogeneous nuclear ribonucleoprotein complex involved in pre-mRNA splicing. The SaGRP protein fluctuates with a very low amplitude over light/dark cycles. Immunogold labeling demonstrates the presence of the SaGRP protein within the nucleus of the investigated meristematic cells of young leaves.

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.

An endogenous timing mechanism, the circadian clock, causes rhythmic expression of a considerable fraction of the genome of most organisms to optimally align physiology and behavior with their environment. Circadian clocks are self-sustained oscillators primarily based on transcriptional feedback loops and post-translational modification of clock proteins. It is increasingly becoming clear that regulation at the RNA level strongly impacts the cellular circadian transcriptome and proteome as well as the oscillator mechanism itself. This review focuses on posttranscriptional events, discussing RNA-binding proteins that, by influencing the timing of pre-mRNA splicing, polyadenylation and RNA decay, shape rhythmic expression profiles. Furthermore, recent findings on the contribution of microRNAs to orchestrating circadian rhythms are summarized.

Plants are highly adapted to respond to a range of environmental stresses commonly by altering their gene expression and metabolism as a result of cell signalling which may be mediated by reactive oxygen species. The glycine-rich RNA-binding proteins AT GRP7 and AT GRP8 were rapidly upregulated in response to peroxide-induced oxidative stress and were amongst the most abundant RNA binding proteins isolated by oligo(dT) chromatography. The oligo(dT)-bound mRNP complexes were analysed proteomically, and were seen to contain potential isoforms of the AT GRP proteins; other proteins that contain an RNA Recognition Motif (RRM); and chloroplast RNA binding proteins. These findings suggest that AT GRP proteins have an evolutionarily conserved function in the regulation of gene expression at the posttranscriptional level in response to environmental stress.

The clock-regulated RNA recognition motif (RRM)-containing protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences the amplitude of its transcript oscillation at the post-transcriptional level. This autoregulation relies on AtGRP7 binding to its own pre-mRNA. The sequence and structural requirements for this interaction are unknown at present. In this work, we used photoinduced electron transfer fluorescence correlation spectroscopy (PET-FCS) as a novel technique to study the role of target RNA secondary structure and conformational dynamics during the recognition and binding process. Conformational dynamics of single-stranded (ss) oligonucleotides were studied in aqueous solution with single-molecule sensitivity and high temporal resolution by monitoring fluorescence quenching of the oxazine fluorophore MR121 by guanosine residues. Comparative analysis of translational diffusion constants revealed that both ssRNA and ssDNA bind to AtGRP7 with similar dissociation constants on the order of 10(-7) M and that a minimal binding sequence 5'-UUC UGG-3' is needed for recognition by AtGRP7. PET-FCS experiments demonstrated that conformational flexibility of short, single-stranded, MR121-labeled oligonucleotides is reduced upon AtGRP7 binding. In contrast to many other RRM proteins, AtGRP7 binds to ssRNA preferentially if the RNA is fully stretched and not embedded within a stable secondary structure. The results suggest that AtGRP7 binding leads to a conformational rearrangement in the mRNA, arresting the flexible target sequence in an extended structure of reduced flexibility that may have consequences for further post-transcriptional processing of the mRNA.

The clock-regulated RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences circadian oscillations of its transcript by negative feedback at the post-transcriptional level. Here we show that site-specific mutation of one conserved arginine to glutamine within the RNA recognition motif impairs binding of recombinant AtGRP7 to its pre-mRNA in vitro. This correlates with the loss of the negative auto-regulation in vivo: in transgenic plants constitutively overexpressing AtGRP7 (AtGRP7-ox), a shift occurs to an alternatively spliced AtGRP7 transcript that decays rapidly, and thus does not accumulate to high levels. In contrast, constitutive ectopic overexpression of the AtGRP7-RQ mutant does not lead to alternative splicing of the endogenous AtGRP7 transcript and concomitant damping of the oscillations. This highlights the importance of AtGRP7 binding to its own transcript for the negative auto-regulatory circuit. Moreover, regulation of AtGRP7 downstream targets also depends on its RNA-binding activity, as AtGRP8 and other targets identified by transcript profiling of wild-type and AtGRP7-ox plants using fluorescent differential display are negatively affected by AtGRP7 but not by AtGRP7-RQ. In mutants impaired in the nonsense-mediated decay (NMD) components UPF1 or UPF3, levels of the alternatively spliced AtGRP7 and AtGRP8 transcripts that contain premature termination codons are strongly elevated, implicating UPF1 and UPF3 in the decay of these clock-regulated transcripts.

The bacterial plant pathogen Pseudomonas syringae injects effector proteins into host cells through a type III protein secretion system to cause disease. The enzymatic activities of most of P. syringae effectors and their targets remain obscure. Here we show that the type III effector HopU1 is a mono-ADP-ribosyltransferase (ADP-RT). HopU1 suppresses plant innate immunity in a manner dependent on its ADP-RT active site. The HopU1 substrates in Arabidopsis thaliana extracts were RNA-binding proteins that possess RNA-recognition motifs (RRMs). A. thaliana knockout lines defective in the glycine-rich RNA-binding protein GRP7 ( also known as AtGRP7), a HopU1 substrate, were more susceptible than wild-type plants to P. syringae. The ADP-ribosylation of GRP7 by HopU1 required two arginines within the RRM, indicating that this modification may interfere with GRP7' s ability to bind RNA. Our results suggest a pathogenic strategy where the ADP-ribosylation of RNA-binding proteins quells host immunity by affecting RNA metabolism and the plant defence transcriptome.

Summary The clock-regulated RNA-binding protein AtGRP7 is part of a negative feedback circuit through which the protein influences circadian oscillations of its own transcript. Constitutive overexpression of AtGRP7 in transgenic plants leads to the appearance of a low amount of an alternatively spliced Atgrp7 transcript with a premature stop codon. It is generated by the use of a 5′ cryptic splice site in the middle of the intron at the expense of the fully spliced mRNA, indicating a role for AtGRP7 in splice site selection. Accelerated decay of this transcript accounts for its low steady state abundance. This implicates a mechanism for the AtGRP7 feedback loop: Atgrp7 expression is downregulated, as AtGRP7 protein accumulates over the circadian cycle, partly by the generation of an alternate transcript that due to its instability does not accumulate to high levels and does not produce a functional protein. Recombinant AtGRP7 protein specifically interacts with the 3′ untranslated region and the intron of its transcript, suggesting that the shift in splice site selection and downregulation involves binding of AtGRP7 to its pre-mRNA. AtGRP7 also influences the choice of splice sites in the Atgrp8 transcript encoding a related RNA-binding protein, favoring the production of an alternatively spliced, unstable Atgrp8 transcript. This conservation points to the importance of this regulatory mechanism to control the level of the clock-regulated glycine-rich RNA-binding proteins and shows how AtGRP7 can control abundance of target transcripts.

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., BMC Plant Biology, 10, ISSN:1471-2229

RNA-binding proteins impact gene expression at the posttranscriptional level by interacting with cognate cis elements within the transcripts. Here, we apply dynamic single-molecule force spectroscopy to study the interaction of the Arabidopsis glycine-rich RNA-binding protein AtGRP8 with its RNA target. A dwell-time-dependent analysis of the single-molecule data in combination with competition assays and site-directed mutagenesis of both the RNA target and the RNA-binding domain of the protein allowed us to distinguish and quantify two different binding modes. For dwell times 0.33 s result in a specific interaction with a lifetime of 208 s. The corresponding reaction lengths are 0.28 nm for the unspecific and 0.55 nm for the specific AtGRP8-RNA interactions, indicating formation of a tighter complex with increasing dwell time. These two binding modes cannot be dissected in ensemble experiments. Quantitative titration in RNA bandshift experiments yields an ensemble-averaged equilibrium constant of dissociation of K-D = 2 x 10(-7) M. Assuming comparable on-rates for the specific and nonspecific binding modes allows us to estimate their free energies as Delta G(0) = -42 kJ/mol and Delta G(0) = -28 kJ/mol for the specific and nonspecific binding modes, respectively. Thus, we show that single-molecule force spectroscopy with a refined statistical analysis is a potent tool for the analysis of protein-RNA interactions without the drawback of ensemble averaging. This makes it possible to discriminate between different binding modes or sites and to analyze them quantitatively. We propose that this method could be applied to complex interactions of biomolecules in general, and be of particular interest for the investigation of multivalent binding reactions.

Alternative splicing (AS) gives rise to multiple mRNA isoforms from the same gene, providing possibilities to regulate gene expression beyond the level of transcription. In a recent paper in Nucleic Acids Research we used a high resolution RT-PCR based panel to study changes in AS patterns in plants with altered levels of an hnRNP-like RNA-binding protein in Arabidopsis thaliana. Furthermore, we detected significant changes in AS patterns between different Arabidopsis ecotypes. Here we investigated how small changes in ambient temperature affect AS. We found significant changes in AS for 12 of 28 investigated events (43%) upon transfer of Arabidopsis plants from 20°C to 16°C and for 6 of the 28 investigated events (21%) upon transfer from 20°C to 24°C.