Genome-wide analysis of mRNA decay in Arabidopsis shoot and root reveals the importance of co-translational mRNA decay in the general mRNA turnover.

Until recently, the general 5'-3' mRNA decay was placed in the cytosol after the mRNA was released from ribosomes. However, the discovery of an additional 5' to 3' pathway, the Co-Translational mRNA Decay (CTRD), changed this paradigm. Up to date, defining the real contribution of CTRD in the general mRNA turnover has been hardly possible as the enzyme involved in this pathway is also involved in cytosolic decay. Here we overcame this obstacle and created an Arabidopsis line specifically impaired for CTRD called XRN4ΔCTRD. Through a genome-wide analysis of mRNA decay rate in shoot and root, we tested the importance of CTRD in mRNA turnover. First, we observed that mRNAs tend to be more stable in root than in shoot. Next, using XRN4ΔCTRD line, we demonstrated that CTRD is a major determinant in mRNA turnover. In shoot, the absence of CTRD leads to the stabilization of thousands of transcripts while in root its absence is highly compensated resulting in faster decay rates. We demonstrated that this faster decay rate is partially due to the XRN4-dependent cytosolic decay. Finally, we correlated this organ-specific effect with XRN4ΔCTRD line phenotypes revealing a crucial role of CTRD in mRNA homeostasis and proper organ development.

Complementary peptides represent a credible alternative to agrochemicals by activating translation of targeted proteins.

The current agriculture main challenge is to maintain food production while facing multiple threats such as increasing world population, temperature increase, lack of agrochemicals due to health issues and uprising of weeds resistant to herbicides. Developing novel, alternative, and safe methods is hence of paramount importance. Here, we show that complementary peptides (cPEPs) from any gene can be designed to target specifically plant coding genes. External application of synthetic peptides increases the abundance of the targeted protein, leading to related phenotypes. Moreover, we provide evidence that cPEPs can be powerful tools in agronomy to improve plant traits, such as growth, resistance to pathogen or heat stress, without the needs of genetic approaches. Finally, by combining their activity they can also be used to reduce weed growth.

Chromatin-associated YTHDC1 coordinates heat-induced reprogramming of gene expression.

Heat stress (HS) induces a cellular response leading to profound changes in gene expression. Here, we show that human YTHDC1, a reader of N6-methyladenosine (m6A) RNA modification, mostly associates to the chromatin fraction and that HS induces a redistribution of YTHDC1 across the genome, including to heat-induced heat shock protein (HSP) genes. YTHDC1 binding to m6A-modified HSP transcripts co-transcriptionally promotes expression of HSPs. In parallel, hundreds of the genes enriched in YTHDC1 during HS have their transcripts undergoing YTHDC1- and m6A-dependent intron retention. Later, YTHDC1 concentrates within nuclear stress bodies (nSBs) where it binds to m6A-modified SATIII non-coding RNAs, produced in an HSF1-dependent manner upon HS. These findings reveal that YTHDC1 plays a central role in a chromatin-associated m6A-based reprogramming of gene expression during HS. Furthermore, they support the model where the subsequent and temporary sequestration of YTHDC1 within nSBs calibrates the timing of this YTHDC1-dependent gene expression reprogramming.

LARP6 proteins in plants.

RNA binding proteins, through control of mRNA fate and expression, are key players of organism development. The LARP family of RBPs sharing the La motif, are largely present in eukaryotes. They classify into five subfamilies which members acquired specific additional domains, including the RRM1 moiety which teams up with the La motif to form a versatile RNA binding unit. The LARP6 subfamily has had a peculiar history during plant evolution. While containing a single LARP6 in algae and non-vascular plants, they expanded and neofunctionalized into three subclusters in vascular plants. Studies from Arabidopsis thaliana, support that they acquired specific RNA binding properties and physiological roles. In particular LARP6C participates, through spatiotemporal control of translation, to male fertilization, a role seemingly conserved in maize. Interestingly, human LARP6 also acts in translation control and mRNA transport and similarly to LARP6C which is required for pollen tube guided elongation, is necessary to cell migration, through protrusion extension. This opens the possibility that some cellular and molecular functions of LARP6 were retained across eukaryote evolution. With their peculiar evolutionary history, plants provide a unique opportunity to uncover how La-module RNA binding properties evolved and identify species specific and basal roles of the LARP6 function. Deciphering of how LARP6, in particular LARP6C, acts at the molecular level, will foster novel knowledge on translation regulation and dynamics in changing cellular contexts. Considering the seemingly conserved function of LARP6C in male reproduction, it should fuel studies aimed at deriving crop species with improved seed yields.

LARP6C orchestrates posttranscriptional reprogramming of gene expression during hydration to promote pollen tube guidance.

Increasing evidence suggests that posttranscriptional regulation is a key player in the transition between mature pollen and the progamic phase (from pollination to fertilization). Nonetheless, the actors in this messenger RNA (mRNA)-based gene expression reprogramming are poorly understood. We demonstrate that the evolutionarily conserved RNA-binding protein LARP6C is necessary for the transition from dry pollen to pollen tubes and the guided growth of pollen tubes towards the ovule in Arabidopsis thaliana. In dry pollen, LARP6C binds to transcripts encoding proteins that function in lipid synthesis and homeostasis, vesicular trafficking, and polarized cell growth. LARP6C also forms cytoplasmic granules that contain the poly(A) binding protein and possibly represent storage sites for translationally silent mRNAs. In pollen tubes, the loss of LARP6C negatively affects the quantities and distribution of storage lipids, as well as vesicular trafficking. In Nicotiana benthamiana leaf cells and in planta, analysis of reporter mRNAs designed from the LARP6C target MGD2 provided evidence that LARP6C can shift from a repressor to an activator of translation when the pollen grain enters the progamic phase. We propose that LARP6C orchestrates the timely posttranscriptional regulation of a subset of mRNAs in pollen during the transition from the quiescent to active state and along the progamic phase to promote male fertilization in plants.

Fast and Efficient 5'P Degradome Library Preparation for Analysis of Co-Translational Decay in Arabidopsis.

The recent development of high-throughput technologies based on RNA sequencing has allowed a better description of the role of post-transcriptional regulation in gene expression. In particular, the development of degradome approaches based on the capture of 5'monophosphate decay intermediates allows the discovery of a new decay pathway called co-translational mRNA decay. Thanks to these approaches, ribosome dynamics could now be revealed by analysis of 5'P reads accumulation. However, library preparation could be difficult to set-up for non-specialists. Here, we present a fast and efficient 5'P degradome library preparation for Arabidopsis samples. Our protocol was designed without commercial kit and gel purification and can be easily done in one working day. We demonstrated the robustness and the reproducibility of our protocol. Finally, we present the bioinformatic reads-outs necessary to assess library quality control.

Characterization of ALBA Family Expression and Localization in Arabidopsis thaliana Generative Organs.

ALBA DNA/RNA-binding proteins form an ancient family, which in eukaryotes diversified into two Rpp25-like and Rpp20-like subfamilies. In most studied model organisms, their function remains unclear, but they are usually associated with RNA metabolism, mRNA translatability and stress response. In plants, the enriched number of ALBA family members remains poorly understood. Here, we studied ALBA dynamics during reproductive development in Arabidopsis at the levels of gene expression and protein localization, both under standard conditions and following heat stress. In generative tissues, ALBA proteins showed the strongest signal in mature pollen where they localized predominantly in cytoplasmic foci, particularly in regions surrounding the vegetative nucleus and sperm cells. Finally, we demonstrated the involvement of two Rpp25-like subfamily members ALBA4 and ALBA6 in RNA metabolism in mature pollen supported by their co-localization with poly(A)-binding protein 3 (PABP3). Collectively, we demonstrated the engagement of ALBA proteins in male reproductive development and the heat stress response, highlighting the involvement of ALBA4 and ALBA6 in RNA metabolism, storage and/or translational control in pollen upon heat stress. Such dynamic re-localization of ALBA proteins in a controlled, developmentally and environmentally regulated manner, likely reflects not only their redundancy but also their possible functional diversification in plants.

Monitoring of XRN4 Targets Reveals the Importance of Cotranslational Decay during Arabidopsis Development.

RNA turnover is a general process that maintains appropriate mRNA abundance at the posttranscriptional level. Although long thought to be antagonistic to translation, discovery of the 5' to 3' cotranslational mRNA decay pathway demonstrated that both processes are intertwined. Cotranslational mRNA decay globally shapes the transcriptome in different organisms and in response to stress; however, the dynamics of this process during plant development is poorly understood. In this study, we used a multiomics approach to reveal the global landscape of cotranslational mRNA decay during Arabidopsis (Arabidopsis thaliana) seedling development. We demonstrated that cotranslational mRNA decay is regulated by developmental cues. Using the EXORIBONUCLEASE4 (XRN4) loss-of-function mutant, we showed that XRN4 poly(A+) mRNA targets are largely subject to cotranslational decay during plant development. As cotranslational mRNA decay is interconnected with translation, we also assessed its role in translation efficiency. We discovered that clusters of transcripts were specifically subjected to cotranslational decay in a developmental-dependent manner to modulate their translation efficiency. Our approach allowed the determination of a cotranslational decay efficiency that could be an alternative to other methods to assess transcript translation efficiency. Thus, our results demonstrate the prevalence of cotranslational mRNA decay in plant development and its role in translational control.

Readers of the m6A epitranscriptomic code.

N6-methyl adenosine (m6A) is the most prevalent and evolutionarily conserved, modification of polymerase II transcribed RNAs. By post-transcriptionally controlling patterns of gene expression, m6A deposition is crucial for organism reproduction, development and likely stress responses. m6A mostly mediates its effect by recruiting reader proteins that either directly accommodate the modified residue in a hydrophobic pocket formed by their YTH domain, or otherwise have their affinity positively influenced by the presence of m6A. We firstly describe here the evolutionary history, and review known molecular and physiological roles of eukaryote YTH readers. In the second part, we present non YTH-proteins whose roles as m6A readers largely remain to be explored. The diversity and multiplicity of m6A readers together with the possibility to regulate their expression and function in response to various cues, offers a multitude of possible combinations to rapidly and finely tune gene expression patterns and hence cellular plasticity. This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.

The YTH Domain Protein ECT2 Is an m6A Reader Required for Normal Trichome Branching in Arabidopsis.

Methylations at position N6 of internal adenosines (m6As) are the most abundant and widespread mRNA modifications. These modifications play crucial roles in reproduction, growth, and development by controlling gene expression patterns at the posttranscriptional level. Their function is decoded by readers that share the YTH domain, which forms a hydrophobic pocket that directly accommodates the m6A residues. While the physiological and molecular functions of YTH readers have been extensively studied in animals, little is known about plant readers, even though m6As are crucial for plant survival and development. Viridiplantae contains high numbers of YTH domain proteins. Here, we performed comprehensive evolutionary analysis of YTH domain proteins and demonstrated that they are highly likely to be actual readers with redundant as well as specific functions. We also show that the ECT2 protein from Arabidopsis thaliana binds to m6A-containing RNAs in vivo and that this property relies on the m6A binding pocket carried by its YTH domain. ECT2 is cytoplasmic and relocates to stress granules upon heat exposure, suggesting that it controls mRNA fate in the cytosol. Finally, we demonstrate that ECT2 acts to decode the m6A signal in the trichome and is required for their normal branching through controlling their ploidy levels.

Positioning Europe for the EPITRANSCRIPTOMICS challenge.

The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.

Heat Shock Protein HSP101 Affects the Release of Ribosomal Protein mRNAs for Recovery after Heat Shock.

Heat shock (HS) is known to have a profound impact on gene expression at different levels, such as inhibition of protein synthesis, in which HS blocks translation initiation and induces the sequestration of mRNAs into stress granules (SGs) or P-bodies for storage and/or decay. SGs prevent the degradation of the stored mRNAs, which can be reengaged into translation in the recovery period. However, little is known on the mRNAs stored during the stress, how these mRNAs are released from SGs afterward, and what the functional importance is of this process. In this work, we report that Arabidopsis HEAT SHOCK PROTEIN101 (HSP101) knockout mutant (hsp101) presented a defect in translation recovery and SG dissociation after HS Using RNA sequencing and RNA immunoprecipitation approaches, we show that mRNAs encoding ribosomal proteins (RPs) were preferentially stored during HS and that these mRNAs were released and translated in an HSP101-dependent manner during recovery. By 15N incorporation and polysome profile analyses, we observed that these released mRNAs contributed to the production of new ribosomes to enhance translation. We propose that, after HS, HSP101 is required for the efficient release of RP mRNAs from SGs resulting in a rapid restoration of the translation machinery by producing new RPs.

Evolutionary history of double-stranded RNA binding proteins in plants: identification of new cofactors involved in easiRNA biogenesis.

In this work, we retrace the evolutionary history of plant double-stranded RNA binding proteins (DRBs), a group of non-catalytic factors containing one or more double-stranded RNA binding motif (dsRBM) that play important roles in small RNA biogenesis and functions. Using a phylogenetic approach, we show that multiple dsRBM DRBs are systematically composed of two different types of dsRBMs evolving under different constraints and likely fulfilling complementary functions. In vascular plants, four distinct clades of multiple dsRBM DRBs are always present with the exception of Brassicaceae species, that do not possess member of the newly identified clade we named DRB6. We also identified a second new and highly conserved DRB family (we named DRB7) whose members possess a single dsRBM that shows concerted evolution with the most C-terminal dsRBM domain of the Dicer-like 4 (DCL4) proteins. Using a BiFC approach, we observed that Arabidopsis thaliana DRB7.2 (AtDRB7.2) can directly interact with AtDRB4 but not with AtDCL4 and we provide evidence that both AtDRB7.2 and AtDRB4 participate in the epigenetically activated siRNAs pathway.

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence.

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. These studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis.

The role of LARP1 in translation and beyond.

The LARP1 proteins form an evolutionarily homogeneous subgroup of the eukaryotic superfamily of La-Motif (LAM) containing factors. Members of the LARP1 family are found in most protists, fungi, plants, and animals. We review here evidence suggesting that LARP1 are key versatile messenger RNA (mRNA)-binding proteins involved in regulating important biological processes such as gametogenesis, embryogenesis, sex determination, and cell division in animals, as well as acclimation to stress in yeasts and plants. LARP1 proteins perform all these essential tasks likely by binding to key mRNAs and regulating their stability and/or translation. In human, the impact of LARP1 over cell division and proliferation is potentially under the control of the TORC1 complex. We review data suggesting that LARP1 is a direct target of this master signaling hub. TOR-dependent LARP1 phosphorylation could specifically enhance the translation of TOP mRNAs providing a way to promote translation, growth, and proliferation. Consequently, LARP1 is found to be significantly upregulated in many malignant cell types. In plants, LARP1 was found to act as a cofactor of the heat-induced mRNA degradation process, an essential acclimation strategy leading to the degradation of more than 4500 mRNAs coding for growth and development housekeeping functions. In Saccharomyces cerevisiae, the LARP1 proteins (Slf1p and Sro9p) are important, among other things, for copper resistance and oxidative stress survival. LARP1 proteins are therefore emerging as critical ancient mRNA-binding factors that evolved common as well as specific targets and regulatory functions in all eukaryotic lineages.

Heat-induced ribosome pausing triggers mRNA co-translational decay in Arabidopsis thaliana.

The reprogramming of gene expression in heat stress is a key determinant to organism survival. Gene expression is downregulated through translation initiation inhibition and release of free mRNPs that are rapidly degraded or stored. In mammals, heat also triggers 5'-ribosome pausing preferentially on transcripts coding for HSC/HSP70 chaperone targets, but the impact of such phenomenon on mRNA fate remains unknown. Here, we provide evidence that, in Arabidopsis thaliana, heat provokes 5'-ribosome pausing leading to the XRN4-mediated 5'-directed decay of translating mRNAs. We also show that hindering HSC/HSP70 activity at 20°C recapitulates heat effects by inducing ribosome pausing and co-translational mRNA turnover. Strikingly, co-translational decay targets encode proteins with high HSC/HSP70 binding scores and hydrophobic N-termini, two characteristics that were previously observed for transcripts most prone to pausing in animals. This work suggests for the first time that stress-induced variation of translation elongation rate is an evolutionarily conserved process leading to the polysomal degradation of thousands of 'non-aberrant' mRNAs.

Parallel action of AtDRB2 and RdDM in the control of transposable element expression.

BACKGROUND: In plants and animals, a large number of double-stranded RNA binding proteins (DRBs) have been shown to act as non-catalytic cofactors of DICERs and to participate in the biogenesis of small RNAs involved in RNA silencing. We have previously shown that the loss of Arabidopsis thaliana's DRB2 protein results in a significant increase in the population of RNA polymerase IV (p4) dependent siRNAs, which are involved in the RNA-directed DNA methylation (RdDM) process. RESULTS: Surprisingly, despite this observation, we show in this work that DRB2 is part of a high molecular weight complex that does not involve RdDM actors but several chromatin regulator proteins, such as MSI4, PRMT4B and HDA19. We show that DRB2 can bind transposable element (TE) transcripts in vivo but that drb2 mutants do not have a significant variation in TE DNA methylation. CONCLUSION: We propose that DRB2 is part of a repressive epigenetic regulator complex involved in a negative feedback loop, adjusting epigenetic state to transcription level at TE loci, in parallel of the RdDM pathway. Loss of DRB2 would mainly result in an increased production of TE transcripts, readily converted in p4-siRNAs by the RdDM machinery.

XRN4 and LARP1 are required for a heat-triggered mRNA decay pathway involved in plant acclimation and survival during thermal stress.

To survive adverse and ever-changing environmental conditions, an organism must be able to adapt. It has long been established that the cellular reaction to stress includes the upregulation of genes coding for specific stress-responsive factors. In the present study, we demonstrate that during the early steps of the heat stress response, 25% of the Arabidopsis seedling transcriptome is targeted for rapid degradation. Our findings demonstrate that this process is catalyzed from 5' to 3' by the cytoplasmic exoribonuclease XRN4, whose function is seemingly reprogrammed by the heat-sensing pathway. The bulk of mRNAs subject to heat-dependent degradation are likely to include both the ribosome-released and polysome associated polyadenylated pools. The cotranslational decay process is facilitated at least in part by LARP1, a heat-specific cofactor of XRN4 required for its targeting to polysomes. Commensurate with their respective involvement at the molecular level, LARP1 and XRN4 are necessary for the thermotolerance of plants to long exposure to moderately high temperature, with xrn4 null mutants being almost unable to survive. These findings provide mechanistic insights regarding a massive stress-induced posttranscriptional downregulation and outline a potentially crucial pathway for plant survival and acclimation to heat stress.