The Arabidopsis tDR Ala forms G-quadruplex structures that can be unwound by the DExH1 DEA(D/H)-box RNA helicase.

As in many other organisms, tRNA-derived RNAs (tDRs) exist in plants and likely have multiple functions. We previously showed that tDRs are present in Arabidopsis under normal growth conditions, and that the ones originating from alanine tRNAs are the most abundant in leaves. We also showed that tDRs Ala of 20 nt produced from mature tRNAAla (AGC) can block in vitro protein translation. Here, we report that first, these tDRs Ala (AGC) can be found within peculiar foci in the cell that are neither P-bodies nor stress granules and, second, that they assemble into intermolecular RNA G-quadruplex (rG4) structures. Such tDR Ala rG4 structures can specifically interact with an Arabidopsis DEA(D/H) RNA helicase, the DExH1 protein, and unwind them. The rG4-DExH1 protein interaction relies on a glycine-arginine domain with RGG/RG/GR/GRR motifs present at the N-terminal extremity of the protein. Mutations on the four guanine residues located at the 5' extremity of the tDR Ala abolish its rG4 structure assembly, association with the DExH1 protein, and foci formation, but they do not prevent protein translation inhibition in vitro. Our data suggest that the sequestration of tDRs Ala into rG4 complexes might represent a way to modulate accessible and functional tDRs for translation inhibition within the plant cell via the activity of a specific RNA helicase, DExH1.

Epigenetic silencing of clustered tRNA genes in Arabidopsis.

Beyond their key role in translation, cytosolic transfer RNAs (tRNAs) are involved in a wide range of other biological processes. Nuclear tRNA genes (tDNAs) are transcribed by the RNA polymerase III (RNAP III) and cis-elements, trans-factors as well as genomic features are known to influence their expression. In Arabidopsis, besides a predominant population of dispersed tDNAs spread along the 5 chromosomes, some clustered tDNAs have been identified. Here, we demonstrate that these tDNA clusters are transcriptionally silent and that pathways involved in the maintenance of DNA methylation play a predominant role in their repression. Moreover, we show that clustered tDNAs exhibit repressive chromatin features whilst their dispersed counterparts contain permissive euchromatic marks. This work demonstrates that both genomic and epigenomic contexts are key players in the regulation of tDNAs transcription. The conservation of most of these regulatory processes suggests that this pioneering work in Arabidopsis can provide new insights into the regulation of RNA Pol III transcription in other organisms, including vertebrates.

Combining tRNA sequencing methods to characterize plant tRNA expression and post-transcriptional modification.

Differences in tRNA expression have been implicated in a remarkable number of biological processes. There is growing evidence that tRNA genes can play dramatically different roles depending on both expression and post-transcriptional modification, yet sequencing tRNAs to measure abundance and detect modifications remains challenging. Their secondary structure and extensive post-transcriptional modifications interfere with RNA-seq library preparation methods and have limited the utility of high-throughput sequencing technologies. Here, we combine two modifications to standard RNA-seq methods by treating with the demethylating enzyme AlkB and ligating with tRNA-specific adapters in order to sequence tRNAs from four species of flowering plants, a group that has been shown to have some of the most extensive rates of post-transcriptional tRNA modifications. This protocol has the advantage of detecting full-length tRNAs and sequence variants that can be used to infer many post-transcriptional modifications. We used the resulting data to produce a modification index of almost all unique reference tRNAs in Arabidopsis thaliana, which exhibited many anciently conserved similarities with humans but also positions that appear to be 'hot spots' for modifications in angiosperm tRNAs. We also found evidence based on northern blot analysis and droplet digital PCR that, even after demethylation treatment, tRNA-seq can produce highly biased estimates of absolute expression levels most likely due to biased reverse transcription. Nevertheless, the generation of full-length tRNA sequences with modification data is still promising for assessing differences in relative tRNA expression across treatments, tissues or subcellular fractions and help elucidate the functional roles of tRNA modifications.

Plant RNases T2, but not Dicer-like proteins, are major players of tRNA-derived fragments biogenesis.

RNA fragments deriving from tRNAs (tRFs) exist in all branches of life and the repertoire of their biological functions regularly increases. Paradoxically, their biogenesis remains unclear. The human RNase A, Angiogenin, and the yeast RNase T2, Rny1p, generate long tRFs after cleavage in the anticodon region. The production of short tRFs after cleavage in the D or T regions is still enigmatic. Here, we show that the Arabidopsis Dicer-like proteins, DCL1-4, do not play a major role in the production of tRFs. Rather, we demonstrate that the Arabidopsis RNases T2, called RNS, are key players of both long and short tRFs biogenesis. Arabidopsis RNS show specific expression profiles. In particular, RNS1 and RNS3 are mainly found in the outer tissues of senescing seeds where they are the main endoribonucleases responsible of tRNA cleavage activity for tRFs production. In plants grown under phosphate starvation conditions, the induction of RNS1 is correlated with the accumulation of specific tRFs. Beyond plants, we also provide evidence that short tRFs can be produced by the yeast Rny1p and that, in vitro, human RNase T2 is also able to generate long and short tRFs. Our data suggest an evolutionary conserved feature of these enzymes in eukaryotes.

The multi-faceted regulation of nuclear tRNA gene transcription.

Transfer RNAs are among the most ancient molecules of life on earth. Beyond their crucial role in protein synthesis as carriers of amino acids, they are also important players in a plethora of other biological processes. Many debates in term of biogenesis, regulation and function persist around these fascinating non-coding RNAs. Our review focuses on the first step of their biogenesis in eukaryotes, i.e. their transcription from nuclear genes. Numerous and complementary ways have emerged during evolution to regulate transfer RNA gene transcription. Here, we will summarize the different actors implicated in this process: cis-elements, trans-factors, genomic contexts, epigenetic environments and finally three-dimensional organization of nuclear genomes. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1099-1108, 2019.

Organization and epigenomic control of RNA polymerase III-transcribed genes in plants.

The genetic information linearly scripted in chromosomes is wrapped in a ribonucleoprotein complex called chromatin. The adaptation of its compaction level and spatiotemporal organization refines gene expression in response to developmental and environmental cues. RNA polymerase III (RNAPIII) is responsible for the biogenesis of elementary non-coding RNAs. Their genes are subjected to high duplication and mutational rates, and invade nuclear genomes. Their insertion into different epigenomic environments raises the question of how chromatin packing affects their individual transcription. In this review, we provide a unique perspective to this issue in plants. In addition, we discuss how the genomic organization of RNAPIII-transcribed loci, combined with epigenetic differences, might participate to plant trait variations.

Spatial Features and Functional Implications of Plant 3D Genome Organization.

The advent of high-throughput sequencing-based methods for chromatin conformation, accessibility, and immunoprecipitation assays has been a turning point in 3D genomics. Altogether, these new tools have been pushing upward the interpretation of pioneer cytogenetic evidence for a higher order in chromatin packing. Here, we review the latest development in our understanding of plant spatial genome structures and different levels of organization and discuss their functional implications. Then, we spotlight the complexity of organellar (i.e., mitochondria and plastids) genomes and discuss their 3D packing into nucleoids. Finally, we propose unaddressed research axes to investigate functional links between chromatin-like dynamics and transcriptional regulation within organellar nucleoids.

Transfection of Small Noncoding RNAs into Arabidopsis thaliana Protoplasts.

Polyethylene glycol transfection of plant protoplasts represents an efficient method to incorporate foreign DNA and study transient gene expression. Here, we describe an optimized protocol to deliver small noncoding RNAs into Arabidopsis thaliana protoplasts. An example of application is provided by demonstrating the incorporation of a 20 nt long small noncoding RNA deriving from the 5' extremity of an A. thaliana cytosolic alanine tRNA into freshly isolated protoplasts.