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.

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.

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.

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.

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

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.

Genome-wide identification and phylogenetic analysis of plant RNA binding proteins comprising both RNA recognition motifs and contiguous glycine residues.

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.

Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy.

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 Kd 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.

VANESA - a software application for the visualization and analysis of networks in system biology applications.

VANESA is a modeling software for the automatic reconstruction and analysis of biological networks based on life-science database information. Using VANESA, scientists are able to model any kind of biological processes and systems as biological networks. It is now possible for scientists to automatically reconstruct important molecular systems with information from the databases KEGG, MINT, IntAct, HPRD, and BRENDA. Additionally, experimental results can be expanded with database information to better analyze the investigated elements and processes in an overall context. Users also have the possibility to use graph theoretical approaches in VANESA to identify regulatory structures and significant actors within the modeled systems. These structures can then be further investigated in the Petri net environment of VANESA. It is platform-independent, free-of-charge, and available at http://vanesa.sf.net.