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.

Cell-type-specific control of secondary cell wall formation by Musashi-type translational regulators in Arabidopsis.

Deciphering the mechanism of secondary cell wall/SCW formation in plants is key to understanding their development and the molecular basis of biomass recalcitrance. Although transcriptional regulation is essential for SCW formation, little is known about the implication of post-transcriptional mechanisms in this process. Here we report that two bonafide RNA-binding proteins homologous to the animal translational regulator Musashi, MSIL2 and MSIL4, function redundantly to control SCW formation in Arabidopsis. MSIL2/4 interactomes are similar and enriched in proteins involved in mRNA binding and translational regulation. MSIL2/4 mutations alter SCW formation in the fibers, leading to a reduction in lignin deposition, and an increase of 4-O-glucuronoxylan methylation. In accordance, quantitative proteomics of stems reveal an overaccumulation of glucuronoxylan biosynthetic machinery, including GXM3, in the msil2/4 mutant stem. We showed that MSIL4 immunoprecipitates GXM mRNAs, suggesting a novel aspect of SCW regulation, linking post-transcriptional control to the regulation of SCW biosynthesis genes.

A High Frequency of Chromosomal Duplications in Unicellular Algae Is Compensated by Translational Regulation.

Although duplications have long been recognized as a fundamental process driving major evolutionary innovations, direct estimates of spontaneous chromosome duplication rates, leading to aneuploid karyotypes, are scarce. Here, from mutation accumulation (MA) experiments, we provide the first estimates of spontaneous chromosome duplication rates in six unicellular eukaryotic species, which range from 1 × 10-4 to 1 × 10-3 per genome per generation. Although this is ∼5 to ∼60 times less frequent than spontaneous point mutations per genome, chromosome duplication events can affect 1-7% of the total genome size. In duplicated chromosomes, mRNA levels reflected gene copy numbers, but the level of translation estimated by polysome profiling revealed that dosage compensation must be occurring. In particular, one duplicated chromosome showed a 2.1-fold increase of mRNA but translation rates were decreased to 0.7-fold. Altogether, our results support previous observations of chromosome-dependent dosage compensation effects, providing evidence that compensation occurs during translation. We hypothesize that an unknown posttranscriptional mechanism modulates the translation of hundreds of transcripts from genes located on duplicated regions in eukaryotes.

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.

Monitoring mRNA Half-Life in Arabidopsis Using Droplet Digital PCR.

mRNA decay is an important process in post-transcriptional regulation; in addition, it plays a crucial role in plant development and response to stress. The development of new tools to quantify mRNA decay intermediates is thus important to better characterize the dynamic of mRNA decay in various conditions. Here, we applied droplet digital PCR (ddPCR), a recent and precise PCR technology, to determine mRNA half-life in Arabidopsis seedlings. We demonstrated that ddPCR can correctly assess mRNA half-life from a wide variety of transcripts in a reproducible manner. We also demonstrated that thanks to multiplexing mRNA, the half-life of multiple transcripts can be followed in the same reaction. As ddPCR allows precise quantification, we proposed that this approach is highly suitable when a low amount of RNA is available; for the detection of many targets or for the analysis of lowly expressed transcripts.

The mRNA-binding proteome of a critical phase transition during Arabidopsis seed germination.

Arabidopsis thaliana seed germination is marked by extensive translational control at two critical phase transitions. The first transition refers to the start of hydration, the hydration translational shift. The second shift, the germination translational shift (GTS) is the phase between testa rupture and radicle protrusion at which the seed makes the all or nothing decision to germinate. The mechanism behind the translational regulation at these phase transitions is unknown. RNA binding proteins (RBPs) are versatile players in the post-transcriptional control of messenger RNAs (mRNAs) and as such candidates for regulating translation during seed germination. Here, we report the mRNA binding protein repertoire of seeds during the GTS. Thirty seed specific RBPs and 22 dynamic RBPs were identified during the GTS, like the putative RBP Vacuolar ATPase subunit A and RBP HSP101. Several stress granule markers were identified in this study, which suggests that seeds are prepared to quickly adapt the translation of specific mRNAs in response to changes in environmental conditions during the GTS. Taken together this study provides a detailed insight into the world of RBPs during seed germination and their possible regulatory role during this developmentally regulated process.

Live single-cell transcriptional dynamics via RNA labelling during the phosphate response in plants.

Plants are constantly adapting to ambient fluctuations through spatial and temporal transcriptional responses. Here, we implemented the latest-generation RNA imaging system and combined it with microfluidics to visualize transcriptional regulation in living Arabidopsis plants. This enabled quantitative measurements of the transcriptional activity of single loci in single cells, in real time and under changing environmental conditions. Using phosphate-responsive genes as a model, we found that active genes displayed high transcription initiation rates (one initiation event every ~3 s) and frequently clustered together in endoreplicated cells. We observed gene bursting and large allelic differences in single cells, revealing that at steady state, intrinsic noise dominated extrinsic variations. Moreover, we established that transcriptional repression triggered in roots by phosphate, a crucial macronutrient limiting plant development, occurred with unexpectedly fast kinetics (on the order of minutes) and striking heterogeneity between neighbouring cells. Access to single-cell RNA polymerase II dynamics in live plants will benefit future studies of signalling processes.

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.

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.

Arabidopsis tRNA-derived fragments as potential modulators of translation.

Transfer RNA-derived fragments (tRFs) exist in all branches of life. They are involved in RNA degradation, regulation of gene expression, ribosome biogenesis. In archaebacteria, kinetoplastid, yeast, and human cells, they were also shown to regulate translation. In Arabidopsis, the tRFs population fluctuates under developmental or environmental conditions but their functions are yet poorly understood. Here, we show that populations of long (30-35 nt) or short (19-25 nt) tRFs produced from Arabidopsis tRNAs can inhibit in vitro translation of a reporter gene. Analysing a series of oligoribonucleotides mimicking natural tRFs, we demonstrate that only a limited set of tRFs possess the ability to affect protein synthesis. Out of a dozen of tRFs, only two deriving from tRNAAla(AGC) and tRNAAsn(GUU) strongly attenuate translation in vitro. Contrary to human tRF(Ala), the 4 Gs present at the 5' extremity of Arabidopsis tRF(Ala) are not implicated in this inhibition while the G18 and G19 residues are essential. Protein synthesis inhibition by tRFs does not require complementarity with the translated mRNA but, having the capability to be associated with polyribosomes, tRFs likely act as general modulation factors of the translation process in plants.

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.

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.

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.

A duplicated NUCLEOLIN gene with antagonistic activity is required for chromatin organization of silent 45S rDNA in Arabidopsis.

In plants as well as in animals, hundreds to thousands of 45S rRNA gene copies localize in Nucleolus Organizer Regions (NORs), and the activation or repression of specific sets of rDNA depends on epigenetic mechanisms. Previously, we reported that the Arabidopsis thaliana nucleolin protein NUC1, an abundant and evolutionarily conserved nucleolar protein in eukaryotic organisms, is required for maintaining DNA methylation levels and for controlling the expression of specific rDNA variants in Arabidopsis. Interestingly, in contrast with animal or yeast cells, plants contain a second nucleolin gene. Here, we report that Arabidopsis NUC1 and NUC2 nucleolin genes are both required for plant growth and survival and that NUC2 disruption represses flowering. However, these genes seem to be functionally antagonistic. In contrast with NUC1, disruption of NUC2 induces CG hypermethylation of rDNA and NOR association with the nucleolus. Moreover, NUC2 loss of function triggers major changes in rDNA spatial organization, expression, and transgenerational stability. Our analyses indicate that silencing of specific rRNA genes is mostly determined by the active or repressed state of the NORs and that nucleolin proteins play a key role in the developmental control of this process.

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.

Uridylation prevents 3' trimming of oligoadenylated mRNAs.

Degradation of mRNAs is usually initiated by deadenylation, the shortening of long poly(A) tails to oligo(A) tails of 12-15 As. Deadenylation leads to decapping and to subsequent 5' to 3' degradation by XRN proteins, or alternatively 3' to 5' degradation by the exosome. Decapping can also be induced by uridylation as shown for the non-polyadenylated histone mRNAs in humans and for several mRNAs in Schizosaccharomyces pombe and Aspergillus nidulans. Here we report a novel role for uridylation in preventing 3' trimming of oligoadenylated mRNAs in Arabidopsis. We show that oligo(A)-tailed mRNAs are uridylated by the cytosolic UTP:RNA uridylyltransferase URT1 and that URT1 has no major impact on mRNA degradation rates. However, in absence of uridylation, oligo(A) tails are trimmed, indicating that uridylation protects oligoadenylated mRNAs from 3' ribonucleolytic attacks. This conclusion is further supported by an increase in 3' truncated transcripts detected in urt1 mutants. We propose that preventing 3' trimming of oligo(A)-tailed mRNAs by uridylation participates in establishing the 5' to 3' directionality of mRNA degradation. Importantly, uridylation prevents 3' shortening of mRNAs associated with polysomes, suggesting that a key biological function of uridylation is to confer 5' to 3' polarity in case of co-translational mRNA decay.

Developmental and environmental regulation of Aquaporin gene expression across Populus species: divergence or redundancy?

Aquaporins (AQPs) are membrane channels belonging to the major intrinsic proteins family and are known for their ability to facilitate water movement. While in Populus trichocarpa, AQP proteins form a large family encompassing fifty-five genes, most of the experimental work focused on a few genes or subfamilies. The current work was undertaken to develop a comprehensive picture of the whole AQP gene family in Populus species by delineating gene expression domain and distinguishing responsiveness to developmental and environmental cues. Since duplication events amplified the poplar AQP family, we addressed the question of expression redundancy between gene duplicates. On these purposes, we carried a meta-analysis of all publicly available Affymetrix experiments. Our in-silico strategy controlled for previously identified biases in cross-species transcriptomics, a necessary step for any comparative transcriptomics based on multispecies design chips. Three poplar AQPs were not supported by any expression data, even in a large collection of situations (abiotic and biotic constraints, temporal oscillations and mutants). The expression of 11 AQPs was never or poorly regulated whatever the wideness of their expression domain and their expression level. Our work highlighted that PtTIP1;4 was the most responsive gene of the AQP family. A high functional divergence between gene duplicates was detected across species and in response to tested cues, except for the root-expressed PtTIP2;3/PtTIP2;4 pair exhibiting 80% convergent responses. Our meta-analysis assessed key features of aquaporin expression which had remained hidden in single experiments, such as expression wideness, response specificity and genotype and environment interactions. By consolidating expression profiles using independent experimental series, we showed that the large expansion of AQP family in poplar was accompanied with a strong divergence of gene expression, even if some cases of functional redundancy could be suspected.

The association of a La module with the PABP-interacting motif PAM2 is a recurrent evolutionary process that led to the neofunctionalization of La-related proteins.

La-related proteins (LARPs) are largely uncharacterized factors, well conserved throughout evolution. Recent reports on the function of human LARP4 and LARP6 suggest that these proteins fulfill key functions in mRNA metabolism and/or translation. We report here a detailed evolutionary history of the LARP4 and 6 families in eukaryotes. Genes coding for LARP4 and 6 were duplicated in the common ancestor of the vertebrate lineage, but one LARP6 gene was subsequently lost in the common ancestor of the eutherian lineage. The LARP6 gene was also independently duplicated several times in the vascular plant lineage. We observed that vertebrate LARP4 and plant LARP6 duplication events were correlated with the acquisition of a PABP-interacting motif 2 (PAM2) and with a significant reorganization of their RNA-binding modules. Using isothermal titration calorimetry (ITC) and immunoprecipitation methods, we show that the two plant PAM2-containing LARP6s (LARP6b and c) can, indeed, interact with the major plant poly(A)-binding protein (PAB2), while the third plant LARP6 (LARP6a) is unable to do so. We also analyzed the RNA-binding properties and the subcellular localizations of the two types of plant LARP6 proteins and found that they display nonredundant characteristics. As a whole, our results support a model in which the acquisition by LARP4 and LARP6 of a PAM2 allowed their targeting to mRNA 3' UTRs and led to their neofunctionalization.