Search

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]

N6‐methyladenosine (m6A) in mRNA is key to eukaryotic gene regulation. Many m6A functions involve RNA‐binding proteins that recognize m6A via a YT521‐B Homology (YTH) domain. YTH domain proteins contain long intrinsically disordered regions (IDRs) that may mediate phase separation and interaction with protein partners, but whose precise biochemical functions remain largely unknown. The Arabidopsis thaliana YTH domain proteins ECT2, ECT3, and ECT4 accelerate organogenesis through stimulation of cell division in organ primordia. Here, we use ECT2 to reveal molecular underpinnings of this function. We show that stimulation of leaf formation requires the long N‐terminal IDR, and we identify two short IDR elements required for ECT2‐mediated organogenesis. Of these two, a 19‐amino acid region containing a tyrosine‐rich motif conserved in both plant and metazoan YTHDF proteins is necessary for binding to the major cytoplasmic poly(A)‐binding proteins PAB2, PAB4, and PAB8. Remarkably, overexpression of PAB4 in leaf primordia partially rescues the delayed leaf formation in ect2 ect3 ect4 mutants, suggesting that the ECT2‐PAB2/4/8 interaction on target mRNAs of organogenesis‐related genes may overcome limiting PAB concentrations in primordial cells. Synopsis: An interaction between m6A‐binding YTHDF proteins and cytoplasmic poly(A)‐binding proteins promotes leaf formation. It is mediated by a short motif conserved across plant and animal YTHDF proteins, suggesting an effector function of m6A readers shared between animals and plants.The intrinsically disordered region of the plant YTHDF protein ECT2 contains a tyrosine‐rich short linear motif (SLiM) required for interaction with the cytoplasmic poly(A)‐binding proteins PAB2, PAB4, and PAB8.This tyrosine‐rich SLiM is required for ECT2‐mediated leaf formation and is deeply conserved in plant and animal YTHDF proteins.Overexpression of the cytoplasmic poly(A)‐binding protein PAB4 in leaf primordia partially rescues the leaf formation defects in mutants carrying knockouts in major YTHDF‐encoding genes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In plants, microRNA (miRNA)-target complementarity has long been considered the predominant factor determining the silencing outcome of the miRNA-target interaction, although the efficacy of such interactions have rarely been appraised in plants. Here, we perform in planta silencing efficacy assays on seven Arabidopsis MYB genes, all of which contain conserved miR159-binding sites of analogous complementarity. These genes were found to be differentially silenced by miR159; MYB81, MYB97, MYB101, MYB104, and DUO1 were all poorly silenced, whereas MYB33 and MYB65 were strongly silenced. Curiously, this is consistent with previous genetic analysis defining MYB33 and MYB65 as the major functional targets of miR159. Neither the free energy of miR159-target complementarity, nor miRNA binding site accessibility, as determined by flanking region AU content, could fully explain the discrepancy of miR159 silencing efficacy. Instead, we found that MYB33 and MYB65 were both predicted to contain a distinctive RNA secondary structure abutting the miR159 binding site. The structure is composed of two stem-loops (SLs) that are predicted to form in MYB33/65 homologs of species as evolutionary distant as gymnosperms. Functional analysis found that the RNA structure in MYB33 correlated with strong silencing efficacy; introducing mutations to disrupt either SL attenuated miR159 efficacy, while introducing complementary mutations to restore the SLs, but not the sequence, restored strong miR159-mediated silencing. Therefore, it appears that this RNA secondary structure demarcates MYB33/65 as sensitive targets of miR159, which underpins the narrow functional specificity of Arabidopsis miR159. [ABSTRACT FROM AUTHOR]

Elucidation of micro RNA (mi RNA) function through a loss-of-function approach has proven difficult due to extensive genetic redundancy among most plant and animal mi RNA families. Consequently, mi RNA decoy technologies such as target MIMICs ( MIMs) and short tandem target MIMICs ( STTMs) in plants or molecular SPONGEs ( SPs) in animals have been developed to generate loss-of-function phenotypes by perturbing endogenous mi RNA activity. To test whether SPs can inhibit plant mi RNA activity, synthetic SP transgenes containing multiple mi RNA binding sites targeting different Arabidopsis mi RNA families were generated. Additionally, their silencing efficacies were compared to the corresponding MIM and STTM transgenes via scoring the frequency and severity of phenotypic abnormalities elicited by each transgene. While SPs with wild-type mi RNA binding sites have no apparent impact, SPs containing mi RNA binding sites with two central mismatches ( cm SPs) can generate strong loss-of-function phenotypes. However, their efficacy varied dramatically, from inducing strong loss-of-function phenotypes to failing to produce any phenotypic impact. Variability was also observed when MIMs and STTMs were compared to cm SPs. While cm SP165/ 166 and STTM165/166 showed a stronger efficacy than MIM165/166, MIM159 was stronger than cm SP159 and STTM159. Although increasing the number of mi RNA binding sites or strengthening the free energy of the mi RNA binding site interaction can improve decoy efficacy, clearly additional unknown overriding factors are at play. In conclusion, we demonstrate that no one approach guarantees the strongest mi RNA inhibition, but rather distinct mi RNA families respond differently to the various approaches, suggesting that multiple approaches may need to be taken to generate the desired loss-of-function 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]

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]

N6-methyladenosine (m 6 A) in mRNA is key to eukaryotic gene regulation. Many m 6 A functions involve RNA-binding proteins that recognize m 6 A via a YT521-B Homology (YTH) domain. YTH domain proteins contain long intrinsically disordered regions (IDRs) that may mediate phase separation and interaction with protein partners, but whose precise biochemical functions remain largely unknown. The Arabidopsis thaliana YTH domain proteins ECT2, ECT3, and ECT4 accelerate organogenesis through stimulation of cell division in organ primordia. Here, we use ECT2 to reveal molecular underpinnings of this function. We show that stimulation of leaf formation requires the long N-terminal IDR, and we identify two short IDR elements required for ECT2-mediated organogenesis. Of these two, a 19-amino acid region containing a tyrosine-rich motif conserved in both plant and metazoan YTHDF proteins is necessary for binding to the major cytoplasmic poly(A)-binding proteins PAB2, PAB4, and PAB8. Remarkably, overexpression of PAB4 in leaf primordia partially rescues the delayed leaf formation in ect2 ect3 ect4 mutants, suggesting that the ECT2-PAB2/4/8 interaction on target mRNAs of organogenesis-related genes may overcome limiting PAB concentrations in primordial cells., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)