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• UV light irradiation is suitable to crosslink RNA and bound proteins in Arabidopsis. • RNA-binding protein targets have been determined by iCLIP, HITS-CLIP and RIP-seq mRNA. • mRNA interactome capture detects many novel plant RNA-binding proteins. 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. [ABSTRACT FROM AUTHOR]

Research into the regulation of gene expression underwent a shift from focusing on DNA-binding proteins as key transcriptional regulators to RNA-binding proteins (RBPs) that come into play once transcription has been initiated. RBPs orchestrate all RNA-processing steps in the cell. To obtain a global view of in vivo targets, the RNA complement associated with particular RBPs is determined via immunoprecipitation of the RBP and subsequent identification of bound RNAs via RNA-seq. Here, we describe technical advances in identifying RBP in vivo targets and their binding motifs. We provide an up-to-date view of targets of nucleocytoplasmic RBPs collected in arabidopsis. We also discuss current experimental limitations and provide an outlook on how the approaches may advance our understanding of post-transcriptional networks. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

n 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. [ABSTRACT FROM AUTHOR]

Die parasitische Pflanze Cuscuta campestris legt in der Wirtspflanze mit Hilfe von microRNAs die Expression von Genen lahm, die der Verteidigung gegen den Parasiten dienen. So kann der Parasit sich an den Nährstoffen der Wirtspflanze bedienen. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

mRNAs und Proteine gehen in der Zelle oft eine Partnerschaft ein. Die neue „mRNA interactome capture“ Methode ermöglicht es, alle Proteine zu erfassen, die in der Zelle an mRNAs gebunden sind. Dazu werden RNA‐bindende Proteine und mRNAs in der Zelle quervernetzt. Isoliert man dann selektiv die mRNAs, fängt man gleichzeitig auch die an mRNAs gebundenen Proteine ein. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: We develop an advanced bioinformatics pipeline to identify RBP targets and their RNA binding motifs from iCLIP data which will be broadly applicable to plant RBP iCLP data. We show that AtGRP7 binds to the LHCB1.1 transcript and decreases its stability with concomitant dampened peak abundance. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m6A) exerts many of its regulatory effects on eukaryotic mRNAs by recruiting cytoplasmic YT521-B homology-domain family (YTHDF) proteins. Here, we show that in Arabidopsis thaliana, the interaction between m6A and the major YTHDF protein ECT2 also involves the mRNA-binding ALBA protein family. ALBA and YTHDF proteins physically associate via a deeply conserved short linear motif in the intrinsically disordered region of YTHDF proteins and their mRNA target sets overlap, with ALBA4 binding sites being juxtaposed to m6A sites. These binding sites correspond to pyrimidine-rich elements previously found to be important for m6A binding to ECT2. Accordingly, both the biological functions of ECT2, and its binding to m6A targets in vivo, require ALBA association. Our results introduce the YTHDF-ALBA complex as the functional cytoplasmic m6A-reader in Arabidopsis, and define a molecular foundation for the concept of facilitated m6A reading, which increases the potential for combinatorial control of biological m6A effects. [ABSTRACT FROM AUTHOR]

Regulation at the RNA level by RNA-binding proteins (RBPs) and microRNAs (miRNAs) is key to coordinating eukaryotic gene expression. In plants, the importance of miRNAs is highlighted by severe developmental defects in mutants impaired in miRNA biogenesis. MiRNAs are processed from long primary-microRNAs (pri-miRNAs) with internal stem-loop structures by endonucleolytic cleavage. The highly structured stem-loops constitute the basis for the extensive regulation of miRNA biogenesis through interaction with RBPs. However, trans-acting regulators of the biogenesis of specific miRNAs are largely unknown in plants. Therefore, we exploit an RNA-centric approach based on modified versions of the conditional CRISPR nuclease Csy4* to pull down interactors of the Arabidopsis pri-miR398b stem-loop (pri-miR398b-SL) in vitro. We designed three epitope-tagged versions of the inactive Csy4* for the immobilization of the protein together with the pri-miR398b-SL bait on high affinity matrices. After incubation with nucleoplasmic extracts from Arabidopsis and extensive washing, pri-miR398b-SL, along with its specifically bound proteins, were released by re-activating the cleavage activity of the Csy4* upon the addition of imidazole. Co-purified proteins were identified via quantitative mass spectrometry and data sets were compared. In total, we identified more than 400 different proteins, of which 180 are co-purified in at least two out of three independent Csy4*-based RNA pulldowns. Among those, the glycine-rich RNA-binding protein AtRZ-1a was identified in all pulldowns. To analyze the role of AtRZ-1a in miRNA biogenesis, we determined the miR398 expression level in the atrz-1a mutant. Indeed, the absence of AtRZ-1a caused a decrease in the steady-state level of mature miR398 with a concomitant reduction in pri-miR398b levels. Overall, we show that our modified Csy4*-based RNA pulldown strategy is suitable to identify new trans-acting regulators of miRNA biogenesis and provides new insights into the post-transcriptional regulation of miRNA processing by plant RBPs. [ABSTRACT FROM AUTHOR]

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 nah G 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 ( Avr Rpt2) 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. [ABSTRACT FROM AUTHOR]

Summary 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 cell fate, 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 reveals post-transcriptional regulators of plant root epidermal cell fate. [ABSTRACT FROM AUTHOR]