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.

ANCHOR: A Technical Approach to Monitor Single-Copy Locus Localization in Planta.

Together with local chromatin structure, gene accessibility, and the presence of transcription factors, gene positioning is implicated in gene expression regulation. Although the basic mechanisms are expected to be conserved in eukaryotes, less is known about the role of gene positioning in plant cells, mainly due to the lack of a highly resolutive approach. In this study, we adapted the use of the ANCHOR system to perform real-time single locus detection in planta. ANCHOR is a DNA-labeling tool derived from the chromosome partitioning system found in many bacterial species. We demonstrated its suitability to monitor a single locus in planta and used this approach to track chromatin mobility during cell differentiation in Arabidopsis thaliana root epidermal cells. Finally, we discussed the potential of this approach to investigate the role of gene positioning during transcription and DNA repair in plants.

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.

Nucleolus-associated chromatin domains are maintained under heat stress, despite nucleolar reorganization in Arabidopsis thaliana.

Several layers of mechanisms participate in plant adaptation to heat-stress. For example, the plant metabolism switches from cell growth mode to stress adaptation mode. Ribosome biogenesis is one of the most energy costly pathways. That biogenesis process occurs in the nucleolus, the largest nuclear compartment, whose structure is highly dependent on this pathway. We used a nucleolar marker to track the structure of the nucleolus, and revealed a change in its sub-nucleolar distribution under heat stress. In addition, the nucleolus is implicated in other cellular processes, such as genome organization within the nucleus. However, our analyses of nucleolus-associated chromatin domains under heat stress did not reveal significant differences compared to the control plants, suggesting a lack of connection between two of the main functions of the nucleolus: ribosome biogenesis and nuclear organization.

The m6A pathway protects the transcriptome integrity by restricting RNA chimera formation in plants.

Global, segmental, and gene duplication-related processes are driving genome size and complexity in plants. Despite their evolutionary potentials, those processes can also have adverse effects on genome regulation, thus implying the existence of specialized corrective mechanisms. Here, we report that an N6-methyladenosine (m6A)-assisted polyadenylation (m-ASP) pathway ensures transcriptome integrity in Arabidopsis thaliana Efficient m-ASP pathway activity requires the m6A methyltransferase-associated factor FIP37 and CPSF30L, an m6A reader corresponding to an YT512-B Homology Domain-containing protein (YTHDC)-type domain containing isoform of the 30-kD subunit of cleavage and polyadenylation specificity factor. Targets of the m-ASP pathway are enriched in recently rearranged gene pairs, displayed an atypical chromatin signature, and showed transcriptional readthrough and mRNA chimera formation in FIP37- and CPSF30L-deficient plants. Furthermore, we showed that the m-ASP pathway can also restrict the formation of chimeric gene/transposable-element transcript, suggesting a possible implication of this pathway in the control of transposable elements at specific locus. Taken together, our results point to selective recognition of 3'-UTR m6A as a safeguard mechanism ensuring transcriptome integrity at rearranged genomic loci in plants.

UAP56 associates with DRM2 and is localized to chromatin in Arabidopsis.

Repeated sequence expression and transposable element mobilization are tightly controlled by multilayer processes, which include DNA 5'-cytosine methylation. The RNA-directed DNA methylation (RdDM) pathway, which uses siRNAs to guide sequence-specific directed DNA methylation, emerged specifically in plants. RdDM ensures DNA methylation maintenance on asymmetric CHH sites and specifically initiates de novo methylation in all cytosine sequence contexts through the action of DRM DNA methyltransferases, of which DRM2 is the most prominent. The RdDM pathway has been well described, but how DRM2 is recruited onto DNA targets and associates with other RdDM factors remains unknown. To address these questions, we developed biochemical approaches to allow the identification of factors that may escape genetic screens, such as proteins encoded by multigenic families. Through both conventional and affinity purification of DRM2, we identified DEAD box RNA helicases U2AF56 Associated Protein 56 (UAP56a/b), which are widespread among eukaryotes, as new DRM2 partners. We have shown that, similar to DRM2 and other RdDM actors, UAP56 has chromatin-associated protein properties. We confirmed this association both in vitro and in vivo in reproductive tissues. In addition, our experiments also suggest that UAP56 may exhibit differential distribution in cells depending on plant organ. While originally identified for its role in splicing, our study suggests that UAP56 may also have other roles, and our findings allow us to initiate discussion about its potential role in the RdDM pathway.

Nucleolar Proteome Analysis and Proteasomal Activity Assays Reveal a Link between Nucleolus and 26S Proteasome in A. thaliana.

In all eukaryotic cells, the nucleolus is functionally and structurally linked to rRNA synthesis and ribosome biogenesis. This compartment contains as well factors involved in other cellular activities, but the functional interconnection between non-ribosomal activities and the nucleolus (structure and function) still remains an open question. Here, we report a novel mass spectrometry analysis of isolated nucleoli from Arabidopsis thaliana plants using the FANoS (Fluorescence Assisted Nucleolus Sorting) strategy. We identified many ribosome biogenesis factors (RBF) and proteins non-related with ribosome biogenesis, in agreement with the recognized multi-functionality of the nucleolus. Interestingly, we found that 26S proteasome subunits localize in the nucleolus and demonstrated that proteasome activity and nucleolus organization are intimately linked to each other. Proteasome subunits form discrete foci in the disorganized nucleolus of nuc1.2 plants. Nuc1.2 protein extracts display reduced proteasome activity in vitro compared to WT protein extracts. Remarkably, proteasome activity in nuc1.2 is similar to proteasome activity in WT plants treated with proteasome inhibitors (MG132 or ALLN). Finally, we show that MG132 treatment induces disruption of nucleolar structures in WT but not in nuc1.2 plants. Altogether, our data suggest a functional interconnection between nucleolus structure and proteasome activity.

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.

Plant nucleolar DNA: Green light shed on the role of Nucleolin in genome organization.

The nucleolus forms as a consequence of ribosome biogenesis, but it is also implicated in other cell functions. The identification of nucleolus-associated chromatin domains (NADs) in animal and plant cells revealed the presence of DNA sequences other than rRNA genes in and around the nucleolus. NADs display repressive chromatin signatures and harbour repetitive DNA, but also tRNA genes and RNA polymerase II-transcribed genes. Furthermore, the identification of NADs revealed a specific function of the nucleolus and the protein Nucleolin 1 (NUC1) in telomere biology. Here, we discuss the significance of these data with regard to nucleolar structure and to the role of the nucleolus and NUC1 in global genome organization and stability.

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.

NERD, a plant-specific GW protein, defines an additional RNAi-dependent chromatin-based pathway in Arabidopsis.

In Arabidopsis, transcriptional gene silencing (TGS) can be triggered by 24 nt small-interfering RNAs (siRNAs) through the RNA-directed DNA methylation (RdDM) pathway. By functional analysis of NERD, a GW repeat- and PHD finger-containing protein, we demonstrate that Arabidopsis harbors a second siRNA-dependent DNA methylation pathway targeting a subset of nonconserved genomic loci. The activity of the NERD-dependent pathway differs from RdDM by the fact that it relies both on silencing-related factors previously implicated only in posttranscriptional gene silencing (PTGS), including RNA-DEPENDENT RNA POLYMERASE1/6 and ARGONAUTE2, and most likely on 21 nt siRNAs. A central role for NERD in integrating RNA silencing and chromatin signals in transcriptional silencing is supported by data showing that it binds both to histone H3 and AGO2 proteins and contributes to siRNA accumulation at a NERD-targeted locus. Our results unravel the existence of a conserved chromatin-based RNA silencing pathway encompassing both PTGS and TGS components in plants.

RNA-directed DNA methylation requires an AGO4-interacting member of the SPT5 elongation factor family.

Recent studies have identified a conserved WG/GW-containing motif, known as the Argonaute (AGO) hook, which is involved in the recruitment of AGOs to distinct components of the eukaryotic RNA silencing pathways. By using this motif as a model to detect new components in plant RNA silencing pathways, we identified SPT5-like, a plant-specific AGO4-interacting member of the nuclear SPT5 (Suppressor of Ty insertion 5) RNA polymerase (RNAP) elongation factor family that is characterized by the presence of a carboxy-terminal extension with more than 40 WG/GW motifs. Knockout SPT5-like mutants show a decrease in the accumulation of several 24-nt RNAs and hypomethylation at different loci revealing an implication in RNA-directed DNA methylation (RdDM). Here, we propose that SPT5-like emerged in plants as a facultative RNAP elongation factor. Its plant-specific origin and role in RdDM might reflect functional interactions with plant-specific RNA Pols required for RdDM.

Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components.

Two forms of RNA Polymerase IV (PolIVa/PolIVb) have been implicated in RNA-directed DNA methylation (RdDM) in Arabidopsis. Prevailing models imply a distinct function for PolIVb by association of Argonaute4 (AGO4) with the C-terminal domain (CTD) of its largest subunit NRPD1b. Here we show that the extended CTD of NRPD1b-type proteins exhibits conserved Argonaute-binding capacity through a WG/GW-rich region that functionally distinguishes Pol IVb from Pol IVa, and that is essential for RdDM. Site-specific mutagenesis and domain-swapping experiments between AtNRPD1b and the human protein GW182 demonstrated that reiterated WG/GW motifs form evolutionarily and functionally conserved Argonaute-binding platforms in RNA interference (RNAi)-related components.