ECT2 peptide sequences outside the YTH domain regulate its m6A-RNA binding.

The m6A epitranscriptomic mark is the most abundant and widespread internal RNA chemical modification, which through the control of RNA acts as an important factor of eukaryote reproduction, growth, morphogenesis and stress response. The main m6A readers constitute a super family of proteins with hundreds of members that share a so-called YTH RNA binding domain. The majority of YTH proteins carry no obvious additional domain except for an Intrinsically Disordered Region (IDR). In Arabidopsis thaliana IDRs are important for the functional specialization among the different YTH proteins, known as Evolutionarily Conserved C-Terminal region, ECT 1 to 12. Here by studying the ECT2 protein and using an in vitro biochemical characterization, we show that full-length ECT2 and its YTH domain alone have a distinct ability to bind m6A, conversely to previously characterized YTH readers. We identify peptide regions outside of ECT2 YTH domain, in the N-terminal IDR, that regulate its binding to m6A-methylated RNA. Furthermore, we show that the selectivity of ECT2 binding for m6A is enhanced by a high uridine content within its neighbouring sequence, where ECT2 N-terminal IDR is believed to contact the target RNA in vivo. Finally, we also identify small structural elements, located next to ECT2 YTH domain and conserved in a large set of YTH proteins, that enhance its binding to m6A-methylated RNA. We propose from these findings that some of these regulatory regions are not limited to ECT2 or YTH readers of flowering plants but may be widespread among eukaryotic YTH readers.

Crosslinking of base-modified RNAs by synthetic DYW-KP base editors implicates an enzymatic lysine as the nitrogen donor for U-to-C RNA editing.

Sequence-specific cytidine to uridine (C-to-U) and adenosine to inosine editing tools can alter RNA and DNA sequences and utilize a hydrolytic deamination mechanism requiring an active site zinc ion and a glutamate residue. In plant organelles, DYW-PG domain containing enzymes catalyze C-to-U edits through the canonical deamination mechanism. Proteins developed from consensus sequences of the related DYW-KP domain family catalyze what initially appeared to be uridine to cytidine (U-to-C) edits leading to this investigation into the U-to-C editing mechanism. The synthetic DYW-KP enzyme KP6 was found sufficient for C-to-U editing activity stimulated by the addition of carboxylic acids in vitro. Despite addition of putative amine/amide donors, U-to-C editing by KP6 could not be observed in vitro. C-to-U editing was found not to be concomitant with U-to-C editing, discounting a pyrimidine transaminase mechanism. RNAs containing base modifications were highly enriched in interphase fractions consistent with covalent crosslinks to KP6, KP2, and KP3 proteins. Mass spectrometry of purified KP2 and KP6 proteins revealed secondary peaks with mass shifts of 319 Da. A U-to-C crosslinking mechanism was projected to explain the link between crosslinking, RNA base changes, and the ∼319 Da mass. In this model, an enzymatic lysine attacks C4 of uridine to form a Schiff base RNA-protein conjugate. Sequenced RT-PCR products from the fern Ceratopteris richardii indicate U-to-C base edits do not preserve proteinaceous crosslinks in planta. Hydrolysis of a protonated Schiff base conjugate releasing cytidine is hypothesized to explain the completed pathway in plants.

PBOX-sRNA-seq uncovers novel features of miRNA modification and identifies selected 5'-tRNA fragments bearing 2'-O-modification.

The concomitant cloning of RNA degradation products is a major concern in standard small RNA-sequencing practices. This not only complicates the characterization of bona fide sRNAs but also hampers cross-batch experimental replicability and sometimes even results in library construction failure. Given that all types of plant canonical small RNAs possess the 3' end 2'-O-methylation modification, a new small RNA sequencing (sRNA-seq) method, designated as PBOX-sRNA-seq, has been developed specifically to capture this modification. PBOX-sRNA-seq, as its name implies, relies on the sequential treatment of RNA samples with phenylboronic acid-polyacrylamide gel electrophoresis (PBA-PAGE) and sodium periodate (NaIO4) oxidation, before sRNA library construction and sequencing. PBOX-sRNA-seq outperformed separate treatments (i.e. PBA-PAGE only or NaIO4 only) in terms of the depletion of unmethylated RNA species and capture 2'-O-modified sRNAs with extra-high purity. Using PBOX-sRNA-seq, we discovered that nascent miRNA-5p/-3p duplexes may undergo mono-cytidylation/uridylation before 2'-O-methylation. We also identified two highly conserved types of 5'-tRNA fragments (tRF) bearing HEN1-independent 2'-O modification (mainly the 13-nt tRF-5aAla and the 26-nt tRF-5bGly). We believe that PBOX-sRNA-seq is powerful for both qualitative and quantitative analyses of sRNAs in plants and piRNAs in animals.

Principles of miRNA/miRNA* function in plant MIRNA processing.

MicroRNAs (miRNAs) are essential regulators of gene expression, defined by their unique biogenesis, which requires the precise excision of the small RNA from an imperfect fold-back precursor. Unlike their animal counterparts, plant miRNA precursors exhibit variations in sizes and shapes. Plant MIRNAs can undergo processing in a base-to-loop or loop-to-base direction, with DICER-LIKE1 (DCL1) releasing the miRNA after two cuts (two-step MIRNAs) or more (sequential MIRNAs). In this study, we demonstrate the critical role of the miRNA/miRNA* duplex region in the processing of miRNA precursors. We observed that endogenous MIRNAs frequently experience suboptimal processing in vivo due to mismatches in the miRNA/miRNA* duplex, a key region that fine-tunes miRNA levels. Enhancing the interaction energy of the miRNA/miRNA* duplex in two-step MIRNAs results in a substantial increase in miRNA levels. Conversely, sequential MIRNAs display distinct and specific requirements for the miRNA/miRNA* duplexes along their foldback structure. Our work establishes a connection between the miRNA/miRNA* structure and precursor processing mechanisms. Furthermore, we reveal a link between the biological function of miRNAs and the processing mechanism of their precursors with the evolution of plant miRNA/miRNA* duplex structures.

The clade-specific target recognition mechanisms of plant RISCs.

Eukaryotic Argonaut proteins (AGOs) assemble RNA-induced silencing complexes (RISCs) with guide RNAs that allow binding to complementary RNA sequences and subsequent silencing of target genes. The model plant Arabidopsis thaliana encodes 10 different AGOs, categorized into three distinct clades based on amino acid sequence similarity. While clade 1 and 2 RISCs are known for their roles in post-transcriptional gene silencing, and clade 3 RISCs are associated with transcriptional gene silencing in the nucleus, the specific mechanisms of how RISCs from each clade recognize their targets remain unclear. In this study, I conducted quantitative binding analyses between RISCs and target nucleic acids with mismatches at various positions, unveiling distinct target binding characteristics unique to each clade. Clade 1 and 2 RISCs require base pairing not only in the seed region but also in the 3' supplementary region for stable target RNA binding, with clade 1 exhibiting a higher stringency. Conversely, clade 3 RISCs tolerate dinucleotide mismatches beyond the seed region. Strikingly, they bind to DNA targets with an affinity equal to or surpassing that of RNA, like prokaryotic AGO complexes. These insights challenge existing views on plant RNA silencing and open avenues for exploring new functions of eukaryotic AGOs.

Molecular basis of A. thaliana KEOPS complex in biosynthesizing tRNA t6A.

In archaea and eukaryotes, the evolutionarily conserved KEOPS is composed of four core subunits-Kae1, Bud32, Cgi121 and Pcc1, and a fifth Gon7/Pcc2 that is found in fungi and metazoa. KEOPS cooperates with Sua5/YRDC to catalyze the biosynthesis of tRNA N6-threonylcarbamoyladenosine (t6A), an essential modification needed for fitness of cellular organisms. Biochemical and structural characterizations of KEOPSs from archaea, yeast and humans have determined a t6A-catalytic role for Kae1 and auxiliary roles for other subunits. However, the precise molecular workings of KEOPSs still remain poorly understood. Here, we investigated the biochemical functions of A. thaliana KEOPS and determined a cryo-EM structure of A. thaliana KEOPS dimer. We show that A. thaliana KEOPS is composed of KAE1, BUD32, CGI121 and PCC1, which adopts a conserved overall arrangement. PCC1 dimerization leads to a KEOPS dimer that is needed for an active t6A-catalytic KEOPS-tRNA assembly. BUD32 participates in direct binding of tRNA to KEOPS and modulates the t6A-catalytic activity of KEOPS via its C-terminal tail and ATP to ADP hydrolysis. CGI121 promotes the binding of tRNA to KEOPS and potentiates the t6A-catalytic activity of KEOPS. These data and findings provide insights into mechanistic understanding of KEOPS machineries.

Synthetic PPR proteins as tools for sequence-specific targeting of RNA.

In native systems, gene expression is regulated by RNA binding proteins. Such proteins have been the target of a great deal of recent research interest, due to the potential for harnessing these regulatory effects for the construction of new biotechnological tools. In particular, focus has been targeted on building synthetic RNA binding proteins for sequence-specific targeting of new RNA transcripts. Pentatricopeptide repeat (PPR) proteins make compelling candidates as synthetic RNA binding proteins to target and bind RNA transcripts of interest, due to their defined RNA binding "code", modular structure, and native capability to deliver catalytic C-terminal domains. In this review, we present a summary of up-to-date understanding of RNA site recognition by PPR proteins, progress towards the design of synthetic PPR proteins for RNA targeting in vitro and in vivo, highlight key areas for further research around these proteins and present an outlook for future applications for synthetic PPR proteins as biotechnological tools.

In vivo single-molecule analysis reveals COOLAIR RNA structural diversity.

Cellular RNAs are heterogeneous with respect to their alternative processing and secondary structures, but the functional importance of this complexity is still poorly understood. A set of alternatively processed antisense non-coding transcripts, which are collectively called COOLAIR, are generated at the Arabidopsis floral-repressor locus FLOWERING LOCUS C (FLC)1. Different isoforms of COOLAIR influence FLC transcriptional output in warm and cold conditions2-7. Here, to further investigate the function of COOLAIR, we developed an RNA structure-profiling method to determine the in vivo structure of single RNA molecules rather than the RNA population average. This revealed that individual isoforms of the COOLAIR transcript adopt multiple structures with different conformational dynamics. The major distally polyadenylated COOLAIR isoform in warm conditions adopts three predominant structural conformations, the proportions and conformations of which change after cold exposure. An alternatively spliced, strongly cold-upregulated distal COOLAIR isoform6 shows high structural diversity, in contrast to proximally polyadenylated COOLAIR. A hyper-variable COOLAIR structural element was identified that was complementary to the FLC transcription start site. Mutations altering the structure of this region changed FLC expression and flowering time, consistent with an important regulatory role of the COOLAIR structure in FLC transcription. Our work demonstrates that isoforms of non-coding RNA transcripts adopt multiple distinct and functionally relevant structural conformations, which change in abundance and shape in response to external conditions.

An optimized method to obtain high-quality RNA from different tissues in Lilium davidii var. unicolor.

The high quality, yield and purity total RNA samples are essential for molecular experiments. However, harvesting high quality RNA in Lilium davidii var. unicolor is a great challenge due to its polysaccharides, polyphenols and other secondary metabolites. In this study, different RNA extraction methods, namely TRIzol method, the modified TRIzol method, Kit method and cetyltrimethylammonium bromide (CTAB) method were employed to obtain total RNA from different tissues in L. davidii var. unicolor. A Nano drop spectrophotometer and 1% agarose gel electrophoresis were used to detect the RNA quality and integrity. Compared with TRIzol, Kit and CTAB methods, the modified TRIzol method obtained higher RNA concentrations from different tissues and the A260/A280 ratios of RNA samples were ranged from 1.97 to 2.27. Thus, the modified TRIzol method was shown to be the most effective RNA extraction protocol in acquiring RNA with high concentrations. Furthermore, the RNA samples isolated by the modified TRIzol and Kit methods were intact, whereas different degrees of degradation happened within RNA samples isolated by the TRIzol and CTAB methods. In addition, the modified TRIzol method could also isolate high-quality RNA from other edible lily bulbs. Taken together, the modified TRIzol method is an efficient method for total RNA isolation from L. davidii var. unicolor.

A Structural Perspective on the RNA Editing of Plant Respiratory Complexes.

The last steps of respiration, a core energy-harvesting process, are carried out by a chain of multi-subunit complexes in the inner mitochondrial membrane. Several essential subunits of the respiratory complexes are RNA-edited in plants, frequently leading to changes in the encoded amino acids. While the impact of RNA editing is clear at the sequence and phenotypic levels, the underlying biochemical explanations for these effects have remained obscure. Here, we used the structures of plant respiratory complex I, complex III2 and complex IV to analyze the impact of the amino acid changes of RNA editing in terms of their location and biochemical features. Through specific examples, we demonstrate how the structural information can explain the phenotypes of RNA-editing mutants. This work shows how the structural perspective can bridge the gap between sequence and phenotype and provides a framework for the continued analysis of RNA-editing mutants in plant mitochondria and, by extension, in chloroplasts.

Wheat in vivo RNA structure landscape reveals a prevalent role of RNA structure in modulating translational subgenome expression asymmetry.

BACKGROUND: Polyploidy, especially allopolyploidy, which entails merging divergent genomes via hybridization and whole-genome duplication (WGD), is a major route to speciation in plants. The duplication among the parental genomes (subgenomes) often leads to one subgenome becoming dominant over the other(s), resulting in subgenome asymmetry in gene content and expression. Polyploid wheats are allopolyploids with most genes present in two (tetraploid) or three (hexaploid) functional copies, which commonly show subgenome expression asymmetry. It is unknown whether a similar subgenome asymmetry exists during translation. We aim to address this key biological question and explore the major contributing factors to subgenome translation asymmetry. RESULTS: Here, we obtain the first tetraploid wheat translatome and reveal that subgenome expression asymmetry exists at the translational level. We further perform in vivo RNA structure profiling to obtain the wheat RNA structure landscape and find that mRNA structure has a strong impact on translation, independent of GC content. We discover a previously uncharacterized contribution of RNA structure in subgenome translation asymmetry. We identify 3564 single-nucleotide variations (SNVs) across the transcriptomes between the two tetraploid wheat subgenomes, which induce large RNA structure disparities. These SNVs are highly conserved within durum wheat cultivars but are divergent in both domesticated and wild emmer wheat. CONCLUSIONS: We successfully determine both the translatome and in vivo RNA structurome in tetraploid wheat. We reveal that RNA structure serves as an important modulator of translational subgenome expression asymmetry in polyploids. Our work provides a new perspective for molecular breeding of major polyploid crops.

NAD-seq for profiling the NAD+ capped transcriptome of Arabidopsis thaliana.

Eukaryotic RNAs can be modified with a non-canonical 5' nicotinamide adenine dinucleotide (NAD+) cap. NAD-seq identifies transcriptome-wide NAD+ capped RNAs. NAD-seq takes advantage of click chemistry to allow the capture of NAD+ capped RNAs. Unlike other approaches, NAD-seq does not require DNA synthesis on beads, but this technique uses full NAD+ capped transcripts eluted from beads as the substrates for strand-specific RNA sequencing library preparation. For complete details on the use and execution of this protocol, please refer to Yu et al. (2021).

Mechanism of siRNA production by a plant Dicer-RNA complex in dicing-competent conformation.

In eukaryotes, small RNAs (sRNAs) play critical roles in multiple biological processes. Dicer endonucleases are a central part of sRNA biogenesis. In plants, DICER-LIKE PROTEIN 3 (DCL3) produces 24-nucleotide (nt) small interfering RNAs (siRNAs) that determine the specificity of the RNA-directed DNA methylation pathway. Here, we determined the structure of a DCL3­pre-siRNA complex in an active dicing-competent state. The 5'-phosphorylated A1 of the guide strand and the 1-nt 3' overhang of the complementary strand are specifically recognized by a positively charged pocket and an aromatic cap, respectively. The 24-nt siRNA length dependence relies on the separation between the 5'-phosphorylated end of the guide RNA and dual cleavage sites formed by the paired ribonuclease III domains. These structural studies, complemented by functional data, provide insight into the dicing principle for Dicers in general.

Arabidopsis thaliana endonuclease V is a ribonuclease specific for inosine-containing single-stranded RNA.

Endonuclease V is highly conserved, both structurally and functionally, from bacteria to humans, and it cleaves the deoxyinosine-containing double-stranded DNA in Escherichia coli, whereas in Homo sapiens it catalyses the inosine-containing single-stranded RNA. Thus, deoxyinosine and inosine are unexpectedly produced by the deamination reactions of adenine in DNA and RNA, respectively. Moreover, adenosine-to-inosine (A-to-I) RNA editing is carried out by adenosine deaminase acting on dsRNA (ADARs). We focused on Arabidopsis thaliana endonuclease V (AtEndoV) activity exhibiting variations in DNA or RNA substrate specificities. Since no ADAR was observed for A-to-I editing in A. thaliana, the possibility of inosine generation by A-to-I editing can be ruled out. Purified AtEndoV protein cleaved the second and third phosphodiester bonds, 3' to inosine in single-strand RNA, at a low reaction temperature of 20-25°C, whereas the AtEndoV (Y100A) protein bearing a mutation in substrate recognition sites did not cleave these bonds. Furthermore, AtEndoV, similar to human EndoV, prefers RNA substrates over DNA substrates, and it could not cleave the inosine-containing double-stranded RNA. Thus, we propose the possibility that AtEndoV functions as an RNA substrate containing inosine induced by RNA damage, and not by A-to-I RNA editing in vivo.

Novel insights into the pervasive role of RNA structure in post-transcriptional regulation of gene expression in plants.

RNA folding is an intrinsic property of RNA that serves a key role in every step of post-transcriptional regulation of gene expression, from RNA maturation to translation in plants. Recent developments of genome-wide RNA structure profiling methods have transformed research in this area enabling focus to shift from individual molecules to the study of tens of thousands of RNAs. Here, we provide a comprehensive review of recent advances in the field. We discuss these new insights of RNA structure functionality within the context of post-transcriptional regulation including mRNA maturation, translation, and RNA degradation in plants. Notably, we also provide an overview of how plants exhibit different RNA structures in response to environmental changes.

Transcriptome profiling in susceptible and tolerant rubber tree clones in response to cassiicolin Cas1, a necrotrophic effector from Corynespora cassiicola.

Corynespora cassiicola, a fungal plant pathogen with a large host range, causes important damages in rubber tree (Hevea brasiliensis), in Asia and Africa. A small secreted protein named cassiicolin was previously identified as a necrotrophic effector required for the virulence of C. cassiicola in specific rubber tree clones. The objective of this study was to decipher the cassiicolin-mediated molecular mechanisms involved in this compatible interaction. We comparatively analyzed the RNA-Seq transcriptomic profiles of leaves treated or not with the purified cassiicolin Cas1, in two rubber clones: PB260 (susceptible) and RRIM600 (tolerant). The reads were mapped against a synthetic transcriptome composed of all available transcriptomic references from the two clones. Genes differentially expressed in response to cassiicolin Cas1 were identified, in each clone, at two different time-points. After de novo annotation of the synthetic transcriptome, we analyzed GO enrichment of the differentially expressed genes in order to elucidate the main functional pathways impacted by cassiicolin. Cassiicolin induced qualitatively similar transcriptional modifications in both the susceptible and the tolerant clones, with a strong negative impact on photosynthesis, and the activation of defense responses via redox signaling, production of pathogenesis-related protein, or activation of the secondary metabolism. In the tolerant clone, transcriptional reprogramming occurred earlier but remained moderate. By contrast, the susceptible clone displayed a late but huge transcriptional burst, characterized by massive induction of phosphorylation events and all the features of a hypersensitive response. These results confirm that cassiicolin Cas1 is a necrotrophic effector triggering a hypersensitive response in susceptible rubber clones, in agreement with the necrotrophic-effector-triggered susceptibility model.

Plant transfer RNA-derived fragments: Biogenesis and functions.

Processing of mature transfer RNAs (tRNAs) produces complex populations of tRNA-derived fragments (tRFs). Emerging evidence shows that tRFs have important functions in bacteria, animals, and plants. Here, we review recent advances in understanding plant tRFs, focusing on their biological and cellular functions, such as regulating stress responses, mediating plant-pathogen interactions, and modulating post-transcriptional gene silencing and translation. We also review sequencing strategies and bioinformatics resources for studying tRFs in plants. Finally, we discuss future directions for plant tRF research, which will expand our knowledge of plant non-coding RNAs.

Evolution of plant telomerase RNAs: farther to the past, deeper to the roots.

The enormous sequence heterogeneity of telomerase RNA (TR) subunits has thus far complicated their characterization in a wider phylogenetic range. Our recent finding that land plant TRs are, similarly to known ciliate TRs, transcribed by RNA polymerase III and under the control of the type-3 promoter, allowed us to design a novel strategy to characterize TRs in early diverging Viridiplantae taxa, as well as in ciliates and other Diaphoretickes lineages. Starting with the characterization of the upstream sequence element of the type 3 promoter that is conserved in a number of small nuclear RNAs, and the expected minimum TR template region as search features, we identified candidate TRs in selected Diaphoretickes genomes. Homologous TRs were then used to build covariance models to identify TRs in more distant species. Transcripts of the identified TRs were confirmed by transcriptomic data, RT-PCR and Northern hybridization. A templating role for one of our candidates was validated in Physcomitrium patens. Analysis of secondary structure demonstrated a deep conservation of motifs (pseudoknot and template boundary element) observed in all published TRs. These results elucidate the evolution of the earliest eukaryotic TRs, linking the common origin of TRs across Diaphoretickes, and underlying evolutionary transitions in telomere repeats.

The transcriptomic response to heat stress of a jujube (Ziziphus jujuba Mill.) cultivar is featured with changed expression of long noncoding RNAs.

Long non-coding RNA (lncRNA) of plant species undergoes dynamic regulation and acts in developmental and stress regulation. Presently, there is little information regarding the identification of lncRNAs in jujube (Ziziphus jujuba Mill.), and it is uncertain whether the lncRNAs could respond to heat stress (HS) or not. In our previous study, a cultivar (Hqing1-HR) of Z. jujuba were treated by HS (45°C) for 0, 1, 3, 5 and 7 days, and it was found that HS globally changed the gene expression by RNA sequencing (RNA-seq) experiments and informatics analyses. In the current study, 8260 lncRNAs were identified successfully from the previous RNA-seq data, and it indicated that lncRNAs expression was also altered globally, suggesting that the lncRNAs might play vital roles in response to HS. Furthermore, bioinformatics analyses of potential target mRNAs of lncRNAs with cis-acting mechanism were performed, and it showed that multiple differentially expressed (DE) mRNAs co-located with DElncRNAs were highly enriched in pathways associated with response to stress and regulation of metabolic process. Taken together, these findings not only provide a comprehensive identification of lncRNAs but also useful clues for molecular mechanism response to HS in jujube.