Revealing the Arabidopsis AtGRP7 mRNA binding proteome by specific enhanced RNA interactome capture.

BACKGROUND: The interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA processing. RNA interactome capture (RIC) techniques have been implemented to catalogue RNA- binding proteins in the cell. In RIC, RNA-protein complexes are stabilized by UV crosslinking in vivo. Polyadenylated RNAs and associated proteins are pulled down from cell lysates using oligo(dT) beads and the RNA-binding proteome is identified by quantitative mass spectrometry. However, insights into the RNA-binding proteome of a single RNA that would yield mechanistic information on how RNA expression patterns are orchestrated, are scarce. RESULTS: Here, we explored RIC in Arabidopsis to identify proteins interacting with a single mRNA, using the circadian clock-regulated Arabidopsis thaliana GLYCINE-RICH RNA-BINDING PROTEIN 7 (AtGRP7) transcript, one of the most abundant transcripts in Arabidopsis, as a showcase. Seedlings were treated with UV light to covalently crosslink RNA and proteins. The AtGRP7 transcript was captured from cell lysates with antisense oligonucleotides directed against the 5'untranslated region (UTR). The efficiency of RNA capture was greatly improved by using locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. Furthermore, performing a tandem capture with two rounds of pulldown with the 5'UTR oligonucleotide increased the yield. In total, we identified 356 proteins enriched relative to a pulldown from atgrp7 mutant plants. These were benchmarked against proteins pulled down from nuclear lysates by AtGRP7 in vitro transcripts immobilized on beads. Among the proteins validated by in vitro interaction we found the family of Acetylation Lowers Binding Affinity (ALBA) proteins. Interaction of ALBA4 with the AtGRP7 RNA was independently validated via individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). The expression of the AtGRP7 transcript in an alba loss-of-function mutant was slightly changed compared to wild-type, demonstrating the functional relevance of the interaction. CONCLUSION: We adapted specific RNA interactome capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up scaling the protocol should be applicable for less abundant transcripts.

Arginine methylation of SM-LIKE PROTEIN 4 antagonistically affects alternative splicing during Arabidopsis stress responses.

Arabidopsis (Arabidopsis thaliana) PROTEIN ARGININE METHYLTRANSFERASE5 (PRMT5) post-translationally modifies RNA-binding proteins by arginine (R) methylation. However, the impact of this modification on the regulation of RNA processing is largely unknown. We used the spliceosome component, SM-LIKE PROTEIN 4 (LSM4), as a paradigm to study the role of R-methylation in RNA processing. We found that LSM4 regulates alternative splicing (AS) of a suite of its in vivo targets identified here. The lsm4 and prmt5 mutants show a considerable overlap of genes with altered AS raising the possibility that splicing of those genes could be regulated by PRMT5-dependent LSM4 methylation. Indeed, LSM4 methylation impacts AS, particularly of genes linked with stress response. Wild-type LSM4 and an unmethylable version complement the lsm4-1 mutant, suggesting that methylation is not critical for growth in normal environments. However, LSM4 methylation increases with abscisic acid and is necessary for plants to grow under abiotic stress. Conversely, bacterial infection reduces LSM4 methylation, and plants that express unmethylable-LSM4 are more resistant to Pseudomonas than those expressing wild-type LSM4. This tolerance correlates with decreased intron retention of immune-response genes upon infection. Taken together, this provides direct evidence that R-methylation adjusts LSM4 function on pre-mRNA splicing in an antagonistic manner in response to biotic and abiotic stress.

Phase separation of GRP7 facilitated by FERONIA-mediated phosphorylation inhibits mRNA translation to modulate plant temperature resilience.

Changes in ambient temperature profoundly affect plant growth and performance. Therefore, the molecular basis of plant acclimation to temperature fluctuation is of great interest. In this study, we discovered that GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7) contributes to cold and heat tolerance in Arabidopsis thaliana. We found that exposure to a warm temperature rapidly induces GRP7 condensates in planta, which can be reversed by transfer to a lower temperature. Cell biology and biochemical assays revealed that GRP7 undergoes liquid-liquid phase separation (LLPS) in vivo and in vitro. LLPS of GRP7 in the cytoplasm contributes to the formation of stress granules that recruit RNA, along with the translation machinery component eukaryotic initiation factor 4E1 (eIF4E1) and the mRNA chaperones COLD SHOCK PROTEIN 1 (CSP1) and CSP3, to inhibit translation. Moreover, natural variations in GRP7 affecting the residue phosphorylated by the receptor kinase FERONIA alter its capacity to undergo LLPS and correlate with the adaptation of some Arabidopsis accessions to a wider temperature range. Taken together, our findings illustrate the role of translational control mediated by GRP7 LLPS to confer plants with temperature resilience.

Mapping protein-RNA binding in plants with individual-nucleotide-resolution UV cross-linking and immunoprecipitation (plant iCLIP2).

Despite crucial roles of RNA-binding proteins (RBPs) in plant physiology and development, methods for determining their transcriptome-wide binding landscape are less developed than those used in other model organisms. Cross-linking and immunoprecipitation (CLIP) methods (based on UV-mediated generation of covalent bonds between RNAs and cognate RBPs in vivo, purification of the cross-linked complexes and identification of the co-purified RNAs by high-throughput sequencing) have been applied mainly in mammalian cells growing in monolayers or in translucent tissue. We have developed plant iCLIP2, an efficient protocol for performing individual-nucleotide-resolution CLIP (iCLIP) in plants, tailored to overcome the experimental hurdles posed by plant tissue. We optimized the UV dosage to efficiently cross-link RNA and proteins in plants and expressed epitope-tagged RBPs under the control of their native promoters in loss-of-function mutants. We select epitopes for which nanobodies are available, allowing stringent conditions for immunopurification of the RNA-protein complexes to be established. To overcome the inherently high RNase content of plant cells, RNase inhibitors are added and the limited RNA fragmentation step is modified. We combine the optimized isolation of RBP-bound RNAs with iCLIP2, a streamlined protocol that greatly enhances the efficiency of library preparation for high-throughput sequencing. Plant researchers with experience in molecular biology and handling of RNA can complete this iCLIP2 protocol in ~5 d. Finally, we describe a bioinformatics workflow to determine targets of Arabidopsis RBPs from iCLIP data, covering all steps from downloading sequencing reads to identifying cross-linking events ( https://github.com/malewins/Plant-iCLIPseq ), and present the R/Bioconductor package BindingSiteFinder to extract reproducible binding sites ( https://bioconductor.org/packages/release/bioc/html/BindingSiteFinder.html ).

Arabidopsis thaliana GLYCINE RICH RNA-BINDING PROTEIN 7 interaction with its iCLIP target LHCB1.1 correlates with changes in RNA stability and circadian oscillation.

The importance of RNA-binding proteins (RBPs) for plant responses to environmental stimuli and development is well documented. Insights into the portfolio of RNAs they recognize, however, clearly lack behind the understanding gathered in non-plant model organisms. Here, we characterize binding of the circadian clock-regulated Arabidopsis thaliana GLYCINE-RICH RNA-BINDING PROTEIN 7 (AtGRP7) to its target transcripts. We identified novel RNA targets from individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) data using an improved bioinformatics pipeline that will be broadly applicable to plant RBP iCLIP data. 2705 transcripts with binding sites were identified in plants expressing AtGRP7-GFP that were not recovered in plants expressing an RNA-binding dead variant or GFP alone. A conserved RNA motif enriched in uridine residues was identified at the AtGRP7 binding sites. NMR titrations confirmed the preference of AtGRP7 for RNAs with a central U-rich motif. Among the bound RNAs, circadian clock-regulated transcripts were overrepresented. Peak abundance of the LHCB1.1 transcript encoding a chlorophyll-binding protein was reduced in plants overexpressing AtGRP7 whereas it was elevated in atgrp7 mutants, indicating that LHCB1.1 was regulated by AtGRP7 in a dose-dependent manner. In plants overexpressing AtGRP7, the LHCB1.1 half-life was shorter compared to wild-type plants whereas in atgrp7 mutant plants, the half-life was significantly longer. Thus, AtGRP7 modulates circadian oscillations of its in vivo binding target LHCB1.1 by affecting RNA stability.

New Horizons in Plant Photoperiodism.

Photoperiod-measuring mechanisms allow organisms to anticipate seasonal changes to align reproduction and growth with appropriate times of the year. This review provides historical and modern context to studies of plant photoperiodism. We describe how studies of photoperiodic flowering in plants led to the first theoretical models of photoperiod-measuring mechanisms in any organism. We discuss how more recent molecular genetic studies in Arabidopsis and rice have revisited these concepts. We then discuss how photoperiod transcriptomics provides new lessons about photoperiodic gene regulatory networks and the discovery of noncanonical photoperiod-measuring systems housed in metabolic networks of plants. This leads to an examination of nonflowering developmental processes controlled by photoperiod, including metabolism and growth. Finally, we highlight the importance of understanding photoperiodism in the context of climate change, delving into the rapid latitudinal migration of plant species and the potential role of photoperiod-measuring systems in generating photic barriers during migration.

HDF1, a novel flowering time regulator identified in a mutant suppressing sensitivity to red light reduced 1 early flowering.

Arabidopsis SENSITIVITY TO RED LIGHT REDUCED 1 (SRR1) delays the transition from vegetative to reproductive development in noninductive conditions. A second-site suppressor screen for novel genes that overcome early flowering of srr1-1 identified a range of suppressor of srr1-1 mutants flowering later than srr1-1 in short photoperiods. Here, we focus on mutants flowering with leaf numbers intermediate between srr1-1 and Col. Ssm67 overcomes srr1-1 early flowering independently of day-length and ambient temperature. Full-genome sequencing and linkage mapping identified a causative SNP in a gene encoding a Haloacid dehalogenase superfamily protein, named HAD-FAMILY REGULATOR OF DEVELOPMENT AND FLOWERING 1 (HDF1). Both, ssm67 and hdf1-1 show increased levels of FLC, indicating that HDF1 is a novel regulator of this floral repressor. HDF1 regulates flowering largely independent of SRR1, as the effect is visible in srr1-1 and in Col, but full activity on FLC may require SRR1. Furthermore, srr1-1 has a delayed leaf initiation rate that is dependent on HDF1, suggesting that SRR1 and HDF1 act together in leaf initiation. Another mutant flowering intermediate between srr1-1 and wt, ssm15, was identified as a new allele of ARABIDOPSIS SUMO PROTEASE 1, previously implicated in the regulation of FLC stability.

Toward a systems view on RNA-binding proteins and associated RNAs in plants: Guilt by association.

RNA-binding proteins (RBPs) have a broad impact on most biochemical, physiological, and developmental processes in a plant's life. RBPs engage in an on-off relationship with their RNA partners, accompanying virtually every stage in RNA processing and function. While the function of a plethora of RBPs in plant development and stress responses has been described, we are lacking a systems-level understanding of components in RNA-based regulation. Novel techniques have substantially enlarged the compendium of proteins with experimental evidence for binding to RNAs in the cell, the RNA-binding proteome. Furthermore, ribonomics methods have been adapted for use in plants to profile the in vivo binding repertoire of RBPs genome-wide. Here, we discuss how recent technological achievements have provided novel insights into the mode of action of plant RBPs at a genome-wide scale. Furthermore, we touch upon two emerging topics, the connection of RBPs to phase separation in the cell and to extracellular RNAs. Finally, we define open questions to be addressed to move toward an integrated understanding of RBP function.

A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis.

BACKGROUND: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. RESULTS: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. CONCLUSIONS: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

Stochastic Variation in DNA Methylation Modulates Nucleosome Occupancy and Alternative Splicing in Arabidopsis thaliana.

Plants use complex gene regulatory mechanisms to overcome diverse environmental challenges. For instance, cold stress induces rapid and massive transcriptome changes via alternative splicing (AS) to confer cold tolerance in plants. In mammals, mounting evidence suggests chromatin structure can regulate co-transcriptional AS. Recent evidence also supports co-transcriptional regulation of AS in plants, but how dynamic changes in DNA methylation and the chromatin structure influence the AS process upon cold stress remains poorly understood. In this study, we used the DNA methylation inhibitor 5-Aza-2'-Deoxycytidine (5-aza-dC) to investigate the role of stochastic variations in DNA methylation and nucleosome occupancy in modulating cold-induced AS, in Arabidopsis thaliana (Arabidopsis). Our results demonstrate that 5-aza-dC derived stochastic hypomethylation modulates nucleosome occupancy and AS profiles of genes implicated in RNA metabolism, plant hormone signal transduction, and of cold-related genes in response to cold stress. We also demonstrate that cold-induced remodelling of DNA methylation regulates genes involved in amino acid metabolism. Collectively, we demonstrate that sudden changes in DNA methylation via drug treatment can influence nucleosome occupancy levels and modulate AS in a temperature-dependent manner to regulate plant metabolism and physiological stress adaptation.

Principles of mRNA targeting via the Arabidopsis m6A-binding protein ECT2.

Specific recognition of N6-methyladenosine (m6A) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The Arabidopsis YTH domain protein ECT2 is thought to bind to mRNA at URU(m6A)Y sites, yet RR(m6A)CH is the canonical m6A consensus site in all eukaryotes and ECT2 functions require m6A-binding activity. Here, we apply iCLIP (individual nucleotide resolution crosslinking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) to define high-quality target sets of ECT2 and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites. Our analyses show that ECT2 does in fact bind to RR(m6A)CH. Pyrimidine-rich motifs are enriched around, but not at m6A sites, reflecting a preference for N6-adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions. Such motifs, particularly oligo-U and UNUNU upstream of m6A sites, are also implicated in ECT2 binding via its intrinsically disordered region (IDR). Finally, URUAY-type motifs are enriched at ECT2 crosslink sites, but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins. Our study provides coherence between genetic and molecular studies of m6A-YTH function in plants and reveals new insight into the mode of RNA recognition by YTH domain-containing proteins.

Genes are strings of genetic code that contain instructions for producing a cell's proteins. Active genes are copied from DNA into molecules called mRNAs, and mRNA molecules are subsequently translated to create new proteins. However, the number of proteins produced by a cell is not only limited by the number of mRNA molecules produced by copying DNA. Cells use a variety of methods to control the stability of mRNA molecules and their translation efficiency to regulate protein production. One of these methods involves adding a chemical tag, a methyl group, onto mRNA while it is being created. These methyl tags can then be used as docking stations by RNA-binding proteins that help regulate protein translation. Most eukaryotic species ­ which include animals, plants and fungi ­ use the same system to add methyl tags to mRNA molecules. One methyl tag in particular, known as m⁶A, is a well-characterised docking site for a particular type of RNA-binding protein that goes by the name of ECT2 in plants. However, in the flowering plant Arabidopsis thaliana, ECT2 was thought to bind to an mRNA sequence different from the one normally carrying the chemical tag, creating obvious confusion about how the system works in plants. Arribas-Hernández, Rennie et al. investigated this question using advanced large-scale biochemical techniques, and discovered that conventional m⁶A methyl tags are indeed used by ECT2 in Arabidopsis thaliana. The confusion likely arose because the sequence ECT2 was thought bind is often located in close proximity to the m⁶A tags, possibly acting as docking stations for proteins that can influence the ability of ECT2 to bind mRNA. Arribas-Hernández, Rennie et al. also uncovered additional mRNA sequences that directly interact with parts of ECT2 previously unknown to participate in mRNA binding. These findings provide new insights into how chemical labels in mRNA control gene activity. They have broad implications that extend beyond plants into other eukaryotic species, including humans. Since this chemical labelling system has a major role in controlling plant growth, these findings could be leveraged in biotechnology applications to improve crop yields and enhance plant-based food production.

RNA-Binding Protein Immunoprecipitation and High-Throughput Sequencing.

The RNA-binding proteome plays a key role in controlling every step in the life of RNA molecules. Through interaction with dedicated sequence motifs, RNA-binding proteins coordinate processing of cohorts of genes. Understanding such posttranscriptional networks controlled by an RNA-binding protein requires a comprehensive identification of its in vivo targets. In Arabidopsis thaliana, RNA immunoprecipitation followed by reverse transcription-PCR has been widely used to test the association of candidate targets with RNA-binding proteins. The detection of unknown target transcripts requires methods operating at the level of the entire transcriptome. Here, we describe a protocol for RNA immunoprecipitation coupled to the generation of libraries from the co-purified RNAs for high-throughput sequencing. This allows determining RNAs associated with RNA-binding proteins in planta at a global scale.

RALF1-FERONIA complex affects splicing dynamics to modulate stress responses and growth in plants.

The environmentally responsive signaling pathways that link global transcriptomic changes through alternative splicing (AS) to plant fitness remain unclear. Here, we found that the interaction of the extracellular rapid alkalinization FACTOR 1 (RALF1) peptide with its receptor FERONIA (FER) triggered a rapid and massive RNA AS response by interacting with and phosphorylating glycine-rich RNA binding protein7 (GRP7) to elevate GRP7 nuclear accumulation in Arabidopsis thaliana. FER-dependent GRP7 phosphorylation enhanced its mRNA binding ability and its association with the spliceosome component U1-70K to enable splice site selection, modulating dynamic AS. Genetic reversal of a RALF1-FER-dependent splicing target partly rescued mutants deficient in GRP7. AS of GRP7 itself induced nonsense-mediated decay feedback to the RALF1-FER-GRP7 module, fine-tuning stress responses, and cell growth. The RALF1-FER-GRP7 module provides a paradigm for regulatory mechanisms of RNA splicing in response to external stimuli.

Plant Individual Nucleotide Resolution Cross-Linking and Immunoprecipitation to Characterize RNA-Protein Complexes.

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.

SEQing: web-based visualization of iCLIP and RNA-seq data in an interactive python framework.

BACKGROUND: RNA-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. RESULTS: Here 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 different 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. CONCLUSION: SEQing 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.

CLIP and RNA interactome studies to unravel genome-wide RNA-protein interactions in vivo in Arabidopsis thaliana.

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.

Different copies of SENSITIVITY TO RED LIGHT REDUCED 1 show strong subfunctionalization in Brassica napus.

BACKGROUND: Correct timing of flowering is critical for plants to produce enough viable offspring. In Arabidopsis thaliana (Arabidopsis), flowering time is regulated by an intricate network of molecular signaling pathways. Arabidopsis srr1-1 mutants lacking SENSITIVITY TO RED LIGHT REDUCED 1 (SRR1) expression flower early, particularly under short day (SD) conditions (1). SRR1 ensures that plants do not flower prematurely in such non-inductive conditions by controlling repression of the key florigen FT. Here, we have examined the role of SRR1 in the closely related crop species Brassica napus. RESULTS: Arabidopsis SRR1 has five homologs in Brassica napus. They can be divided into two groups, where the A02 and C02 copies show high similarity to AtSRR1 on the protein level. The other group, including the A03, A10 and C09 copies all carry a larger deletion in the amino acid sequence. Three of the homologs are expressed at detectable levels: A02, C02 and C09. Notably, the gene copies show a differential expression pattern between spring and winter type accessions of B. napus. When the three expressed gene copies were introduced into the srr1-1 background, only A02 and C02 were able to complement the srr1-1 early flowering phenotype, while C09 could not. Transcriptional analysis of known SRR1 targets in Bna.SRR1-transformed lines showed that CYCLING DOF FACTOR 1 (CDF1) expression is key for flowering time control via SRR1. CONCLUSIONS: We observed subfunctionalization of the B. napus SRR1 gene copies, with differential expression between early and late flowering accessions of some Bna.SRR1 copies. This suggests involvement of Bna.SRR1 in regulation of seasonal flowering in B. napus. The C09 gene copy was unable to complement srr1-1 plants, but is highly expressed in B. napus, suggesting specialization of a particular function. Furthermore, the C09 protein carries a deletion which may pinpoint a key region of the SRR1 protein potentially important for its molecular function. This is important evidence of functional domain annotation in the highly conserved but unique SRR1 amino acid sequence.

Marking RNA: m6A writers, readers, and functions in Arabidopsis.

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 have 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 A. thaliana. We highlight similarities and differences to m6A modification in mammals and provide an outlook on important questions that remain to be addressed.

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-ß. 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.