RNA-binding Protein AtGRP7 Research Articles

Glycine-Rich RNA-Binding Protein AtGRP7 Functions in Nickel and Lead Tolerance in Arabidopsis.

Plant glycine-rich RNA-binding proteins (GRPs) play crucial roles in the response to environmental stresses. However, the functions of AtGRP7 in plants under heavy metal stress remain unclear. In the present study, in Arabidopsis, the transcript level of AtGRP7 was markedly increased by Ni but was decreased by Pb. AtGRP7-overexpressing plants improved Ni tolerance, whereas the knockout mutant (grp7) was more susceptible than the wild type to Ni. In addition, grp7 showed greatly enhanced Pb tolerance, whereas overexpression lines showed high Pb sensitivity. Ni accumulation was reduced in overexpression lines but increased in grp7, whereas Pb accumulation in grp7 was lower than that in overexpression lines. Ni induced glutathione synthase genes GS1 and GS2 in overexpression lines, whereas Pb increased metallothionein genes MT4a and MT4b and phytochelatin synthase genes PCS1 and PCS2 in grp7. Furthermore, Ni increased CuSOD1 and GR1 in grp7, whereas Pb significantly induced FeSOD1 and FeSOD2 in overexpression lines. The mRNA stability of GS2 and PCS1 was directly regulated by AtGRP7 under Ni and Pb, respectively. Collectively, these results indicate that AtGRP7 plays a crucial role in Ni and Pb tolerance by reducing Ni and Pb accumulation and the direct or indirect post-transcriptional regulation of genes related to heavy metal chelators and antioxidant enzymes.

Regulation of Flowering Time by the RNA-Binding Proteins AtGRP7 and AtGRP8

The timing of floral initiation is a tightly controlled process in plants. The circadian clock regulated glycine-rich RNA-binding protein (RBP) AtGRP7, a known regulator of splicing, was previously shown to regulate flowering time mainly by affecting the MADS-box repressor FLOWERING LOCUS C (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. RNA interference- mediated reduction of the level of the paralogous AtGRP8 in atgrp7-1 further delays floral transition compared of with atgrp7-1. AtGRP7 acts in parallel to FCA, FPA and FLK in the branch of the autonomous pathway (AP) comprised of RBPs. It acts in the same branch as FLOWERING LOCUS D, and AtGRP7 loss-of-function mutants show elevated levels of dimethylated lysine 4 of histone H3, a mark for active transcription. In addition to its role in the AP, AtGRP7 acts in the thermosensory pathway of flowering time control by regulating alternative splicing of the floral repressor FLOWERING LOCUS M (FLM). Overexpression of AtGRP7 selectively favors the formation of the repressive isoform FLM-β. Our results suggest that the RBPs AtGRP7 and AtGRP8 influence MADS-Box transcription factors in at least two different pathways of flowering time control. This highlights the importance of RBPs 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.

Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7

BackgroundFunctions 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.ResultsiCLIP 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.ConclusionsWe 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.

Regulation of pri-miRNA processing by the hnRNP-like protein AtGRP7 in Arabidopsis.

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.

Expression of Arabidopsis glycine-rich RNA-binding protein AtGRP2 or AtGRP7 improves grain yield of rice (Oryza sativa) under drought stress conditions

Although posttranscriptional regulation of RNA metabolism is increasingly recognized as a key regulatory process in plant response to environmental stresses, reports demonstrating the importance of RNA metabolism control in crop improvement under adverse environmental stresses are severely limited. To investigate the potential use of RNA-binding proteins (RBPs) in developing stress-tolerant transgenic crops, we generated transgenic rice plants (Oryza sativa) that express Arabidopsis thaliana glycine-rich RBP (AtGRP) 2 or 7, which have been determined to harbor RNA chaperone activity and confer stress tolerance in Arabidopsis, and analyzed the response of the transgenic rice plants to abiotic stresses. AtGRP2- or AtGRP7-expressing transgenic rice plants displayed similar phenotypes comparable with the wild-type plants under high salt or cold stress conditions. By contrast, AtGRP2- or AtGRP7-expressing transgenic rice plants showed much higher recovery rates and grain yields compared with the wild-type plants under drought stress conditions. The higher grain yield of the transgenic rice plants was due to the increases in filled grain numbers per panicle. Collectively, the present results show the importance of posttranscriptional regulation of RNA metabolism in plant response to environmental stress and suggest that GRPs can be utilized to improve the yield potential of crops under stress conditions.

Salicylic acid‐dependent and ‐independent impact of an RNA‐binding protein on plant immunity

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.

An hnRNP-like RNA-binding protein affects alternative splicing by in vivo interaction with transcripts in Arabidopsis thaliana

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 small glycine‐rich RNA binding protein AtGRP7 promotes floral transition in Arabidopsis thaliana

The RNA binding protein AtGRP7 is part of a circadian slave oscillator in Arabidopsis thaliana that negatively autoregulates its own mRNA, and affects the levels of other transcripts. Here, we identify a novel role for AtGRP7 as a flowering-time gene. An atgrp7-1 T-DNA mutant flowers later than wild-type plants under both long and short days, and independent RNA interference lines with reduced levels of AtGRP7, and the closely related AtGRP8 protein, are also late flowering, particularly in short photoperiods. Consistent with the retention of a photoperiodic response, the transcript encoding the key photoperiodic regulator CONSTANS oscillates with a similar pattern in atgrp7-1 and wild-type plants. In both the RNAi lines and in the atgrp7-1 mutant transcript levels for the floral repressor FLC are elevated. Conversely, in transgenic plants ectopically overexpressing AtGRP7, the transition to flowering is accelerated mainly in short days, with a concomitant reduction in FLC abundance. The late-flowering phenotype of the RNAi lines is suppressed by introducing the flc-3 loss-of-function mutation, suggesting that AtGRP7 promotes floral transition, at least partly by downregulating FLC. Furthermore, vernalization overrides the late-flowering phenotype. Retention of both the photoperiodic response and vernalization response are features of autonomous pathway mutants, suggesting that AtGRP7 is a novel member of the autonomous pathway.

Arabidopsis transportin1 is the nuclear import receptor for the circadian clock-regulated RNA-binding protein AtGRP7.

We characterized the Arabidopsis orthologue of the human nuclear import receptor transportin1 (TRN1). Like the human receptor, Arabidopsis TRN1 recognizes nuclear import signals on proteins that are different from the classical basic nuclear localization signals. The M9 domain of human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is the prototype of such signals. We show that AtTRN1 binds to similar domains in hnRNP-like proteins from plants. AtTRN1 also interacts with human hnRNP A1 and with yeast Nab2p, two classical import cargo proteins of transportin in these organisms. Like all nuclear transport receptors of the importin-beta family, AtTRN1 binds to the regulatory GTPase Ran from Arabidopsis. We demonstrated that the amino terminus of AtTRN1 is necessary for this interaction. Recombinant AtTRN1 conferred nuclear import of fluorescently labelled BSA-M9 peptide conjugates in permeabilized HeLa cells, functionally replacing human TRN1 in these in vitro nuclear import assays. We identified three plant substrate proteins that interact with AtTRN1 and contain M9-like domains: a novel Arabidopsis hnRNP that shows high similarity to human hnRNP A1 and two small RNA-binding proteins from Arabidopsis, AtGRP7 and AtGRP8. Nuclear import activity of the M9-like domains of these plant proteins was demonstrated in vivo by their ability to confer partial nuclear re-localisation of a GFP fusion protein containing a nuclear export signal. In addition, fluorescently labelled AtGRP7 was specifically imported into nuclei of permeabilized HeLa cells by Arabidopsis AtTRN1 and human TRN1. These results suggest that the transportin-mediated nuclear import pathway is highly conserved between man, yeast and plants.

The circadian clock regulated RNA-binding protein AtGRP7 autoregulates its expression by influencing alternative splicing of its own pre-mRNA.

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.

RNA-binding proteins and circadian rhythms in Arabidopsis thaliana.

An Arabidopsis transcript preferentially expressed at the end of the daily light period codes for the RNA-binding protein AtGRP7. A reverse genetic approach in Arabidopsis thaliana has 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. The AtGPR7 protein accumulates with a certain delay and represses further accumulation of its transcript, presumably at the post-transcriptional level. In this respect, the AtGRP7 feedback loop differs from known circadian oscillators in the fruitfly Drosophila and mammals based on oscillating clock proteins that repress transcription of their own genes with a 24 h rhythm. It is proposed that the AtGRP7 feedback loop may act within an output pathway from the Arabidopsis clock.