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.

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.

ECT2 peptide sequences outside the YTH domain regulate its m6A-RNA binding.

The m6A epitranscriptomic mark is the most abundant and widespread internal RNA chemical modification, which through the control of RNA acts as an important factor of eukaryote reproduction, growth, morphogenesis and stress response. The main m6A readers constitute a super family of proteins with hundreds of members that share a so-called YTH RNA binding domain. The majority of YTH proteins carry no obvious additional domain except for an Intrinsically Disordered Region (IDR). In Arabidopsis thaliana IDRs are important for the functional specialization among the different YTH proteins, known as Evolutionarily Conserved C-Terminal region, ECT 1 to 12. Here by studying the ECT2 protein and using an in vitro biochemical characterization, we show that full-length ECT2 and its YTH domain alone have a distinct ability to bind m6A, conversely to previously characterized YTH readers. We identify peptide regions outside of ECT2 YTH domain, in the N-terminal IDR, that regulate its binding to m6A-methylated RNA. Furthermore, we show that the selectivity of ECT2 binding for m6A is enhanced by a high uridine content within its neighbouring sequence, where ECT2 N-terminal IDR is believed to contact the target RNA in vivo. Finally, we also identify small structural elements, located next to ECT2 YTH domain and conserved in a large set of YTH proteins, that enhance its binding to m6A-methylated RNA. We propose from these findings that some of these regulatory regions are not limited to ECT2 or YTH readers of flowering plants but may be widespread among eukaryotic YTH readers.

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.

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.

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.

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.

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.

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.

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.