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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 )., (© 2024. Springer Nature Limited.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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]

Plant microRNAs (miRNAs) are critical regulators of gene expression, however little attention has been given to the principles governing miRNA silencing efficacy. Here, we utilize the highly conserved Arabidopsis miR159-MYB33/MYB65 regulatory module to explore these principles. Firstly, we show that perfect central complementarity is not required for strong silencing. Artificial miR159 variants with two cleavage site mismatches can potently silence MYB33/MYB65, fully complementing a loss-of-function mir159 mutant. Moreover, these miR159 variants can cleave MYB33/MYB65 mRNA, however cleavage appears attenuated, as the ratio of cleavage products to full length transcripts decreases with increasing central mismatches. Nevertheless, high levels of un-cleaved MYB33/MYB65 transcripts are strongly silenced by a non-cleavage mechanism. Contrary to MIR159a variants that strongly silenced endogenous MYB33/MYB65, artificial MYB33 variants with central mismatches to miR159 are not efficiently silenced. We demonstrate that differences in the miRNA:target mRNA stoichiometry underlie this paradox. Increasing miR159 abundance in the MYB33 variants results in a strong silencing outcome, whereas increasing MYB33 transcript levels in the MIR159a variants results in a poor silencing outcome. Finally, we identify highly conserved nucleotides that flank the miR159 binding site in MYB33, and demonstrate that they are critical for efficient silencing, as mutation of these flanking nucleotides attenuates silencing at a level similar to that of central mismatches. This implies that the context in which the miRNA binding site resides is a key determinant in controlling the degree of silencing and that a miRNA “target site” encompasses sequences that extend beyond the miRNA binding site. In conclusion, our findings dismiss the notion that miRNA:target complementarity, underpinned by central matches, is the sole dictator of the silencing outcome. [ABSTRACT FROM AUTHOR]

Plant microRNAs (miRNAs) are critical regulators of gene expression, however little attention has been given to the principles governing miRNA silencing efficacy. Here, we utilize the highly conserved Arabidopsis miR159-MYB33/MYB65 regulatory module to explore these principles. Firstly, we show that perfect central complementarity is not required for strong silencing. Artificial miR159 variants with two cleavage site mismatches can potently silence MYB33/MYB65, fully complementing a loss-of-function mir159 mutant. Moreover, these miR159 variants can cleave MYB33/MYB65 mRNA, however cleavage appears attenuated, as the ratio of cleavage products to full length transcripts decreases with increasing central mismatches. Nevertheless, high levels of un-cleaved MYB33/MYB65 transcripts are strongly silenced by a non-cleavage mechanism. Contrary to MIR159a variants that strongly silenced endogenous MYB33/MYB65, artificial MYB33 variants with central mismatches to miR159 are not efficiently silenced. We demonstrate that differences in the miRNA:target mRNA stoichiometry underlie this paradox. Increasing miR159 abundance in the MYB33 variants results in a strong silencing outcome, whereas increasing MYB33 transcript levels in the MIR159a variants results in a poor silencing outcome. Finally, we identify highly conserved nucleotides that flank the miR159 binding site in MYB33, and demonstrate that they are critical for efficient silencing, as mutation of these flanking nucleotides attenuates silencing at a level similar to that of central mismatches. This implies that the context in which the miRNA binding site resides is a key determinant in controlling the degree of silencing and that a miRNA “target site” encompasses sequences that extend beyond the miRNA binding site. In conclusion, our findings dismiss the notion that miRNA:target complementarity, underpinned by central matches, is the sole dictator of the silencing outcome. [ABSTRACT FROM AUTHOR]

Plants and animals microRNAs (miRNAs) have been proposed to be key regulators of many fundamental processes. However defining miRNAs function has been problematic due to the paucity of miRNA loss-of-function mutants. This is likely due to their small gene size and redundancy as most miRNA have highly related family members. Consequently, the analysis of miRNA function has been primarily based on predictive bioinformatic or transgenic gain-of-function approaches. However, a number of new methodologies have been developed able to result in loss-of-function phenotypes. This includes miRNA sponges in animals and target mimicry in plants, both of which sequesters the mature miRNAs, disrupting endogenous miRNA:mRNA target relationships. Furthermore, artificial miRNAs and RNA interference in plants have been shown to be potent silencers of MIRNA genes. We will discuss the strengths and weaknesses of these methodologies which are potentially of great biotechnological use in medicine and agriculture. [ABSTRACT FROM AUTHOR]

The circadian clock is an important endogenous timekeeper, helping plants to prepare for the periodic changes of light and darkness in their environment. The clockwork of this molecular timer is made up of clock proteins that regulate transcription of their own genes with a 24 h rhythm. Furthermore, the rhythmically expressed clock proteins regulate time-of-day dependent transcription of downstream genes, causing messenger RNA (mRNA) oscillations of a large part of the transcriptome. On top of the transcriptional regulation by the clock, circadian rhythms in mRNAs rely in large parts on post-transcriptional regulation, including alternative pre-mRNA splicing, mRNA degradation, and translational control. Here, we present recent insights into the contribution of post-transcriptional regulation to core clock function and to regulation of circadian gene expression in Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]