Neurodevelopmental disorders associated variants in ADAT3 disrupt the activity of the ADAT2/ADAT3 tRNA deaminase complex and impair neuronal migration.

The ADAT2/ADAT3 complex catalyzes the adenosine to inosine modification at the wobble position of eukaryotic tRNAs. Mutations in ADAT3 , the catalytically inactive subunit of the ADAT2/ADAT3 complex, have been identified in patients presenting with severe neurodevelopmental disorders (NDDs). Yet, the physiological function of ADAT2/ADAT3 complex during brain development remains totally unknown. Here we showed that maintaining a proper level of ADAT2/ADAT3 catalytic activity is required for correct radial migration of projection neurons in the developing mouse cortex. In addition, we not only reported 7 new NDD patients carrying biallelic variants in ADAT3 but also deeply characterize the impact of those variants on ADAT2/ADAT3 structure, biochemical properties, enzymatic activity and tRNAs editing and abundance. We demonstrated that all the identified variants alter both the expression and the activity of the complex leading to a significant decrease of I 34 with direct consequence on their steady-state. Using in vivo complementation assays, we correlated the severity of the migration phenotype with the degree of the loss of function caused by the variants. Altogether, our results indicate a critical role of ADAT2/ADAT3 during cortical development and provide cellular and molecular insights into the pathogenicity of ADAT3-related neurodevelopmental disorder.

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.

The decoupled evolution of the organellar genomes of Silene nutans leads to distinct roles in the speciation process.

There is growing evidence that cytonuclear incompatibilities (i.e. disruption of cytonuclear coadaptation) might contribute to the speciation process. In a former study, we described the possible involvement of plastid-nuclear incompatibilities in the reproductive isolation between four lineages of Silene nutans (Caryophyllaceae). Because organellar genomes are usually cotransmitted, we assessed whether the mitochondrial genome could also be involved in the speciation process, knowing that the gynodioecious breeding system of S. nutans is expected to impact the evolutionary dynamics of this genome. Using hybrid capture and high-throughput DNA sequencing, we analyzed diversity patterns in the genic content of the organellar genomes in the four S. nutans lineages. Contrary to the plastid genome, which exhibited a large number of fixed substitutions between lineages, extensive sharing of polymorphisms between lineages was found in the mitochondrial genome. In addition, numerous recombination-like events were detected in the mitochondrial genome, loosening the linkage disequilibrium between the organellar genomes and leading to decoupled evolution. These results suggest that gynodioecy shaped mitochondrial diversity through balancing selection, maintaining ancestral polymorphism and, thus, limiting the involvement of the mitochondrial genome in evolution of hybrid inviability between S. nutans lineages.

Plant tRNA functions beyond their major role in translation.

Transfer RNAs (tRNAs) are well known for their essential function as adapters in delivering amino acids to ribosomes and making the link between mRNA and protein according to the genetic code. Besides this central role in protein synthesis, other functions are attributed to these macromolecules, or their genes, in all living organisms. This review focuses on these extra functions of tRNAs in photosynthetic organisms. For example, tRNAs are implicated in tetrapyrrole biosynthesis, mRNA stabilization or transport, and priming the reverse transcription of viral RNAs, and tRNA-like structures play important roles in RNA viral genomes. Another important function of tRNAs in regulating gene expression is related to their cleavage allowing the production of small non-coding RNAs termed tRNA-derived RNAs. Here, we examine in more detail the biogenesis of tRNA-derived RNAs and their emerging functions in plants.

PlantRNA 2.0: an updated database dedicated to tRNAs of photosynthetic eukaryotes.

PlantRNA (http://plantrna.ibmp.cnrs.fr/) is a comprehensive database of transfer RNA (tRNA) gene sequences retrieved from fully annotated nuclear, plastidial and mitochondrial genomes of photosynthetic organisms. In the first release (PlantRNA 1.0), tRNA genes from 11 organisms were annotated. In this second version, the annotation was implemented to 51 photosynthetic species covering the whole phylogenetic tree of photosynthetic organisms, from the most basal group of Archeplastida, the glaucophyte Cyanophora paradoxa, to various land plants. tRNA genes from lower photosynthetic organisms such as streptophyte algae or lycophytes as well as extremophile photosynthetic species such as Eutrema parvulum were incorporated in the database. As a whole, about 37 000 tRNA genes were accurately annotated. In the frame of the tRNA genes annotation from the genome of the Rhodophyte Chondrus crispus, non-canonical splicing sites in the D- or T-regions of tRNA molecules were identified and experimentally validated. As for PlantRNA 1.0, comprehensive biological information including 5'- and 3'-flanking sequences, A and B box sequences, region of transcription initiation and poly(T) transcription termination stretches, tRNA intron sequences and tRNA mitochondrial import are included.

How to build a ribosome from RNA fragments in Chlamydomonas mitochondria.

Mitochondria are the powerhouse of eukaryotic cells. They possess their own gene expression machineries where highly divergent and specialized ribosomes, named hereafter mitoribosomes, translate the few essential messenger RNAs still encoded by mitochondrial genomes. Here, we present a biochemical and structural characterization of the mitoribosome in the model green alga Chlamydomonas reinhardtii, as well as a functional study of some of its specific components. Single particle cryo-electron microscopy resolves how the Chlamydomonas mitoribosome is assembled from 13 rRNA fragments encoded by separate non-contiguous gene pieces. Additional proteins, mainly OPR, PPR and mTERF helical repeat proteins, are found in Chlamydomonas mitoribosome, revealing the structure of an OPR protein in complex with its RNA binding partner. Targeted amiRNA silencing indicates that these ribosomal proteins are required for mitoribosome integrity. Finally, we use cryo-electron tomography to show that Chlamydomonas mitoribosomes are attached to the inner mitochondrial membrane via two contact points mediated by Chlamydomonas-specific proteins. Our study expands our understanding of mitoribosome diversity and the various strategies these specialized molecular machines adopt for membrane tethering.

Rapid Shifts in Mitochondrial tRNA Import in a Plant Lineage with Extensive Mitochondrial tRNA Gene Loss.

In most eukaryotes, transfer RNAs (tRNAs) are one of the very few classes of genes remaining in the mitochondrial genome, but some mitochondria have lost these vestiges of their prokaryotic ancestry. Sequencing of mitogenomes from the flowering plant genus Silene previously revealed a large range in tRNA gene content, suggesting rapid and ongoing gene loss/replacement. Here, we use this system to test longstanding hypotheses about how mitochondrial tRNA genes are replaced by importing nuclear-encoded tRNAs. We traced the evolutionary history of these gene loss events by sequencing mitochondrial genomes from key outgroups (Agrostemma githago and Silene [=Lychnis] chalcedonica). We then performed the first global sequencing of purified plant mitochondrial tRNA populations to characterize the expression of mitochondrial-encoded tRNAs and the identity of imported nuclear-encoded tRNAs. We also confirmed the utility of high-throughput sequencing methods for the detection of tRNA import by sequencing mitochondrial tRNA populations in a species (Solanum tuberosum) with known tRNA trafficking patterns. Mitochondrial tRNA sequencing in Silene revealed substantial shifts in the abundance of some nuclear-encoded tRNAs in conjunction with their recent history of mt-tRNA gene loss and surprising cases where tRNAs with anticodons still encoded in the mitochondrial genome also appeared to be imported. These data suggest that nuclear-encoded counterparts are likely replacing mitochondrial tRNAs even in systems with recent mitochondrial tRNA gene loss, and the redundant import of a nuclear-encoded tRNA may provide a mechanism for functional replacement between translation systems separated by billions of years of evolutionary divergence.

The structure of the mouse ADAT2/ADAT3 complex reveals the molecular basis for mammalian tRNA wobble adenosine-to-inosine deamination.

Post-transcriptional modification of tRNA wobble adenosine into inosine is crucial for decoding multiple mRNA codons by a single tRNA. The eukaryotic wobble adenosine-to-inosine modification is catalysed by the ADAT (ADAT2/ADAT3) complex that modifies up to eight tRNAs, requiring a full tRNA for activity. Yet, ADAT catalytic mechanism and its implication in neurodevelopmental disorders remain poorly understood. Here, we have characterized mouse ADAT and provide the molecular basis for tRNAs deamination by ADAT2 as well as ADAT3 inactivation by loss of catalytic and tRNA-binding determinants. We show that tRNA binding and deamination can vary depending on the cognate tRNA but absolutely rely on the eukaryote-specific ADAT3 N-terminal domain. This domain can rotate with respect to the ADAT catalytic domain to present and position the tRNA anticodon-stem-loop correctly in ADAT2 active site. A founder mutation in the ADAT3 N-terminal domain, which causes intellectual disability, does not affect tRNA binding despite the structural changes it induces but most likely hinders optimal presentation of the tRNA anticodon-stem-loop to ADAT2.

Cyanophora paradoxa mitochondrial tRNAs play a double game.

Present-day mitochondria derive from a single endosymbiosis of an α-proteobacterium into a proto-eukaryotic cell. Since this monophyletic event, mitochondria have evolved considerably, and unique traits have been independently acquired in the different eukaryotic kingdoms. Mitochondrial genome expression and RNA metabolism have diverged greatly. Here, Cyanophora paradoxa, a freshwater alga considered as a living fossil among photosynthetic organisms, represents an exciting model for studying the evolution of mitochondrial gene expression. As expected, fully mature tRNAs are released from primary transcripts to function in mitochondrial translation. We also show that these tRNAs take part in an mRNA processing punctuation mechanism in a non-conventional manner, leading to mRNA-tRNA hybrids with a CCA triplet at their 3'-extremities. In this case, tRNAs are probably used as stabilizing structures impeding the degradation of mRNA by exonucleases. From our data we propose that the present-day tRNA-like elements (t-elements) found at the 3'-terminals of mitochondrial mRNAs in land plants originate from true tRNAs like those observed in the mitochondria of this basal photosynthetic glaucophyte.

Synthetic biological circuit tested in spaceflight.

Synthetic biology has potential spaceflight applications yet few if any studies have attempted to translate Earth-based synthetic biology tools into spaceflight. An exogenously inducible biological circuit for protein production in Arabidopsis thaliana, pX7-AtPDSi (Guo et al. 2003), was flown to ISS and functionally investigated. Seedlings were grown in a custom built 1.25 U plant greenhouse. Images recorded during the experiment show that leaves of pX7-AtPDSi seedlings photobleached as designed while wild type Col-0 leaves did not, which reveals that the synthetic circuit led to protein production during spaceflight. Polymerase chain reaction analysis post-flight also confirms that the Cre/LoxP (recombination system) portions of the circuit were functional in spaceflight. The subcomponents of the biological circuit, estrogen-responsive transcription factor XVE, Cre/LoxP DNA recombination system, and RNAi post-transcriptional gene silencing system now have flight heritage and can be incorporated in future designs for space applications. To facilitate future plant studies in space, the full payload design and manufacturing files are made available.

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.

La Société de Biologie de Strasbourg : 100 ans au service de la science et de la société.

Founded in 1919, the Society of Biology of Strasbourg (SBS) is a learned society whose purpose is the dissemination and promotion of scientific knowledge in biology. Subsidiary of the Society of Biology, the SBS celebrated its Centenary on Wednesday, the 16th of October 2019 on the Strasbourg University campus and at the Strasbourg City Hall. This day allowed retracing the various milestones of the SBS, through its main strengths, its difficulties and its permanent goal to meet scientific and societal challenges. The common thread of this day was the transmission of knowledge related to the past, the present, but also the future. At the start of the 21st century, the SBS must continue to reinvent itself to pursue its objective of transmitting scientific knowledge in biology and beyond. Scientific talks performed by senior scientists and former SBS thesis prizes awardees, a round table, and informal discussions reflected the history and the dynamism of the SBS association. All SBS Centennial participants have set the first milestone for the SBS Bicentennial.

TITLE: La Société de Biologie de Strasbourg : 100 ans au service de la science et de la société. ABSTRACT: Filiale de la Société de Biologie, la Société de Biologie de Strasbourg (SBS) est une société savante qui a pour objet la diffusion et la promotion du savoir scientifique en biologie et en médecine. Fondée en 1919, La SBS a célébré son Centenaire le mercredi 16 octobre 2019. Cette journée a permis de retracer les différents jalons de la SBS, à travers ses lignes de forces, ses difficultés et sa volonté permanente de mettre en exergue les défis scientifiques et sociétaux auxquels participent les recherches strasbourgeoises. Le fil rouge de cette journée a été la transmission d'un savoir en lien avec le passé, le présent, mais également le futur. En ce début du 21e siècle, la SBS se doit de continuer de se réinventer pour poursuivre son objectif de transmission des connaissances scientifiques en biologie et au-delà. L'ensemble des participants du Centenaire de la SBS a ainsi posé la première pierre du Bicentenaire de la SBS.

Alanine tRNAs Translate Environment Into Behavior in Caenorhabditis elegans.

Caenorhabditis elegans nematodes produce and maintain imprints of attractive chemosensory cues to which they are exposed early in life. Early odor-exposure increases adult chemo-attraction to the same cues. Imprinting is transiently or stably inherited, depending on the number of exposed generations. We show here that the Alanine tRNA (UGC) plays a central role in regulating C. elegans chemo-attraction. Naive worms fed on tRNAAla (UGC) purified from odor-experienced worms, acquire odor-specific imprints. Chemo-attractive responses require the tRNA-modifying Elongator complex sub-units 1 (elpc-1) and 3 (elpc-3) genes. elpc-3 deletions impair chemo-attraction, which is fully restored by wild-type tRNAAla (UGC) feeding. A stably inherited decrease of odor-specific responses ensues from early odor-exposition of elpc-1 deletion mutants. tRNAAla (UGC) may adopt various chemical forms to mediate the cross-talk between innately-programmed and environment-directed chemo-attractive behavior.

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.

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.

Arabidopsis Voltage-Dependent Anion Channels (VDACs): Overlapping and Specific Functions in Mitochondria.

Voltage-dependent anion channels (VDACs) are essential components of the mitochondrial outer membrane. VDACs are involved in the exchange of numerous ions and molecules, from ATP to larger molecules such as tRNAs, and are supposed to adjust exchanges in response to cell signals and stresses. Four major VDACs have been identified in Arabidopsis thaliana. The goal of this study was to explore the specific functions of these proteins, in particular, in tRNA import into mitochondria and stress response. The main results were: (i) VDACs appeared to differentially interact with tRNAs, and VDAC4 could be the major tRNA channel on the outer membrane, (ii) a VDAC3 mRNA isoform was found induced by different stresses, suggesting that VDAC3 might be specifically involved in early steps of stress response and (iii) an analysis of vdac3 and vdac1 mutant lines showed that VDAC3 and VDAC1 shared some, but not all functions. In conclusion, this work brings new knowledge on VDACs, which do not appear as interchangeable pores of the outer membrane and each VDAC has its own specificity.