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).

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.

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.

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.

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.

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.

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.

Selenium and anthocyanins share the same transcription factors R2R3MYB and bHLH in wheat.

Anthocyanins and selenium have vital biological functions for human and plants, they were investigated thoroughly and separately in plants. Previous studies indicated pigmented fruits and vegetables had higher selenium concentration, but whether there is a relationship between anthocyanins and selenium is unclear. In this study, a combined phenotypic and genotypic methodological approach was undertaken to explore the potential relationship between anthocyanins and selenium accumulation by using phenotypic investigation and RNA-seq analysis. The results showed that pigmented cultivars enrichment in Se is a general phenomenon observed for these tested species, this due to pigmented cultivars have higher Se efficiency absorption. Se flow direction mainly improve concentration of S-rich proteins of LMW-GS. This may be a result of the MYB and bHLH co-regulate anthocyanins biosynthesis and Se metabolism at the transcriptional level. This thesis addresses a neglected aspect of the relevant relationship between anthocyanins and selenium.

MIR172d Is Required for Floral Organ Identity and Number in Tomato.

MicroRNA172 (miR172) functions as a central regulator of flowering time and flower development by post-transcriptional repression of APETALA2-LIKE transcription factors. In the model crop Solanum lycopersicum (tomato), the miR172 family is still poorly annotated and information about the functions of specific members is lacking. Here, de-novo prediction of tomato miR172 coding loci identified seven genes (SlMIR172a-g), that code for four unique species of miR172 (sly-miR172). During reproductive development, sly-miR172s are differentially expressed, with sly-miR172c and sly-miR172d being the most abundant members in developing flowers, and are predicted to guide the cleavage of eight APETALA2-LIKE transcription factors. By CRISPR-Cas9 co-targeting of SlMIR172c and SlMIR172d we have generated a battery of loss-of-function and hypomorphic mutants (slmir172c-dCR). The slmir172c-dCR plants developed normal shoot but their flowers displayed graded floral organ abnormalities. Whereas slmir172cCR loss-of-function caused only a slight greening of petals and stamens, hypomorphic and loss-of-function slmir172dCR alleles were associated with the conversion of petals and stamens to sepaloids, which were produced in excess. Interestingly, the degrees of floral organ identity alteration and proliferation were directly correlated with the reduction in sly-miR172d activity. These results suggest that sly-miR172d regulates in a dose-dependent manner floral organ identity and number, likely by negatively regulating its APETALA2-like targets.

R-loop resolution promotes co-transcriptional chromatin silencing.

RNA-mediated chromatin silencing is central to genome regulation in many organisms. However, how nascent non-coding transcripts regulate chromatin is poorly understood. Here, through analysis of Arabidopsis FLC, we show that resolution of a nascent-transcript-induced R-loop promotes chromatin silencing. Stabilization of an antisense-induced R-loop at the 3' end of FLC enables an RNA binding protein FCA, with its direct partner FY/WDR33 and other 3'-end processing factors, to polyadenylate the nascent antisense transcript. This clears the R-loop and recruits the chromatin modifiers demethylating H3K4me1. FCA immunoprecipitates with components of the m6A writer complex, and m6A modification affects dynamics of FCA nuclear condensates, and promotes FLC chromatin silencing. This mechanism also targets other loci in the Arabidopsis genome, and consistent with this fca and fy are hypersensitive to a DNA damage-inducing drug. These results show how modulation of R-loop stability by co-transcriptional RNA processing can trigger chromatin silencing.

Transcriptome analysis reveals defense-related genes and pathways against Xanthomonas campestris pv. vesicatoria in pepper (Capsicum annuum L.).

Bacterial spot (BS), incited by Xanthomonas campestris pv. vesicatoria (Xcv), is one of the most serious diseases of pepper. For a comparative analysis of defense responses to Xcv infection, we performed a transcriptomic analysis of a susceptible cultivar, ECW, and a resistant cultivar, VI037601, using the HiSeqTM 2500 sequencing platform. Approximately 120.23 G clean bases were generated from 18 libraries. From the libraries generated, a total of 38,269 expressed genes containing 11,714 novel genes and 11,232 differentially expressed genes (DEGs) were identified. Functional enrichment analysis revealed that the most noticeable pathways were plant-pathogen interaction, MAPK signaling pathway-plant, plant hormone signal transduction and secondary metabolisms. 1,599 potentially defense-related genes linked to pattern recognition receptors (PRRs), mitogen-activated protein kinase (MAPK), calcium signaling, and transcription factors may regulate pepper resistance to Xcv. Moreover, after Xcv inoculation, 364 DEGs differentially expressed only in VI037601 and 852 genes in both ECW and VI037601. Many of those genes were classified as NBS-LRR genes, oxidoreductase gene, WRKY and NAC transcription factors, and they were mainly involved in metabolic process, response to stimulus and biological regulation pathways. Quantitative RT-PCR of sixteen selected DEGs further validated the RNA-seq differential gene expression analysis. Our results will provide a valuable resource for understanding the molecular mechanisms of pepper resistance to Xcv infection and improving pepper resistance cultivars against Xcv.

RNA methylation in chloroplasts or mitochondria in plants.

Recent advances in our understanding of epitranscriptomic RNA methylation have expanded the complexity of gene expression regulation beyond epigenetic regulation involving DNA methylation and histone modifications. The instalment, removal, and interpretation of methylation marks on RNAs are carried out by writers (methyltransferases), erasers (demethylases), and readers (RNA-binding proteins), respectively. Contrary to an emerging body of evidence demonstrating the importance of RNA methylation in the diverse fates of RNA molecules, including splicing, export, translation, and decay in the nucleus and cytoplasm, their roles in plant organelles remain largely unclear and are only now being discovered. In particular, extremely high levels of methylation marks in chloroplast and mitochondrial RNAs suggest that RNA methylation plays essential roles in organellar biogenesis and functions in plants that are crucial for plant development and responses to environmental stimuli. Thus, unveiling the cellular components involved in RNA methylation in cell organelles is essential to better understand plant biology.

Extensive profiling of the expressions of tRNAs and tRNA-derived fragments (tRFs) reveals the complexities of tRNA and tRF populations in plants.

Evidence is emerging that tRNA-derived fragments (tRFs) are regulatory molecules. Studies of tRFs in plants have been based on conventional small RNA sequencing, and focused on profiling of tRF-5 and tRF-3 species. A more comprehensive and quantitative analysis of the entire tRF population is highly necessary. Here, we employ tRNA-seq and YAMAT-seq, and develop a bioinformatics tool to comprehensively profile the expressions of tRNAs and tRFs in plants. We show that in Arabidopsis, approximately half of tRNA genes are extremely weakly expressed, accounting for only 1% of total tRNA abundance, while ~12% of tRNA genes contribute to ~80% of tRNA abundance. Our tRNA sequencings in various plants reveal that tRNA expression profiles exhibit a cross-species conserved pattern. By characterizing the composition of a highly heterogeneous tRF population, we show that tRNA halves and previously unnoticed 10-16-nt tiny tRFs represent substantial portions. The highly accumulated 13-nt and 16-nt tiny tRFs in Arabidopsis indicate that tiny tRFs are not random tRNA degradation products. Finally, we provide a user-friendly database for displaying the dynamic spatiotemporal expressions of tRNAs and tRFs in the model plants Arabidopsis and rice.