Promoter-pervasive transcription causes RNA polymerase II pausing to boost DOG1 expression in response to salt.

Eukaryotic genomes are pervasively transcribed by RNA polymerase II. Yet, the molecular and biological implications of such a phenomenon are still largely puzzling. Here, we describe noncoding RNA transcription upstream of the Arabidopsis thaliana DOG1 gene, which governs salt stress responses and is a key regulator of seed dormancy. We find that expression of the DOG1 gene is induced by salt stress, thereby causing a delay in seed germination. We uncover extensive transcriptional activity on the promoter of the DOG1 gene, which produces a variety of lncRNAs. These lncRNAs, named PUPPIES, are co-directionally transcribed and extend into the DOG1 coding region. We show that PUPPIES RNAs respond to salt stress and boost DOG1 expression, resulting in delayed germination. This positive role of pervasive PUPPIES transcription on DOG1 gene expression is associated with augmented pausing of RNA polymerase II, slower transcription and higher transcriptional burst size. These findings highlight the positive role of upstream co-directional transcription in controlling transcriptional dynamics of downstream genes.

Light Regulates Plant Alternative Splicing through the Control of Transcriptional Elongation.

Light makes carbon fixation possible, allowing plant and animal life on Earth. We have previously shown that light regulates alternative splicing in plants. Light initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing of a subset of Arabidopsis thaliana transcripts. Here, we show that light promotes RNA polymerase II (Pol II) elongation in the affected genes, whereas in darkness, elongation is lower. These changes in transcription are consistent with elongation causing the observed changes in alternative splicing, as revealed by different drug treatments and genetic evidence. The light control of splicing and elongation is abolished in an Arabidopsis mutant defective in the transcription factor IIS (TFIIS). We report that the chloroplast control of nuclear alternative splicing in plants responds to the kinetic coupling mechanism found in mammalian cells, providing unique evidence that coupling is important for a whole organism to respond to environmental cues.

The nucleolar protein NOL12 is required for processing of large ribosomal subunit rRNA precursors in Arabidopsis.

BACKGROUND: NOL12 5'-3' exoribonucleases, conserved among eukaryotes, play important roles in pre-rRNA processing, ribosome assembly and export. The most well-described yeast counterpart, Rrp17, is required for maturation of 5.8 and 25S rRNAs, whereas human hNOL12 is crucial for the separation of the large (LSU) and small (SSU) ribosome subunit rRNA precursors. RESULTS: In this study we demonstrate that plant AtNOL12 is also involved in rRNA biogenesis, specifically in the processing of the LSU rRNA precursor, 27S pre-rRNA. Importantly, the absence of AtNOL12 alters the expression of many ribosomal protein and ribosome biogenesis genes. These changes could potentially exacerbate rRNA biogenesis defects, or, conversely, they might stem from the disturbed ribosome assembly caused by delayed pre-rRNA processing. Moreover, exposure of the nol12 mutant to stress factors, including heat and pathogen Pseudomonas syringae, enhances the observed molecular phenotypes, linking pre-rRNA processing to stress response pathways. The aberrant rRNA processing, dependent on AtNOL12, could impact ribosome function, as suggested by improved mutant resistance to ribosome-targeting antibiotics. CONCLUSION: Despite extensive studies, the pre-rRNA processing pathway in plants remains insufficiently characterized. Our investigation reveals the involvement of AtNOL12 in the maturation of rRNA precursors, correlating this process to stress response in Arabidopsis. These findings contribute to a more comprehensive understanding of plant ribosome biogenesis.

Single seeds exhibit transcriptional heterogeneity during secondary dormancy induction.

Seeds are highly resilient to the external environment, which allows plants to persist in unpredictable and unfavorable conditions. Some plant species have adopted a bet-hedging strategy to germinate a variable fraction of seeds in any given condition, and this could be explained by population-based threshold models. Here, in the model plant Arabidopsis (Arabidopsis thaliana), we induced secondary dormancy (SD) to address the transcriptional heterogeneity among seeds that leads to binary germination/nongermination outcomes. We developed a single-seed RNA-seq strategy that allowed us to observe a reduction in seed transcriptional heterogeneity as seeds enter stress conditions, followed by an increase during recovery. We identified groups of genes whose expression showed a specific pattern through a time course and used these groups to position the individual seeds along the transcriptional gradient of germination competence. In agreement, transcriptomes of dormancy-deficient seeds (mutant of DELAY OF GERMINATION 1) showed a shift toward higher values of the germination competence index. Interestingly, a significant fraction of genes with variable expression encoded translation-related factors. In summary, interrogating hundreds of single-seed transcriptomes during SD-inducing treatment revealed variability among the transcriptomes that could result from the distribution of population-based sensitivity thresholds. Our results also showed that single-seed RNA-seq is the method of choice for analyzing seed bet-hedging-related phenomena.

Arabidopsis DXO1 links RNA turnover and chloroplast function independently of its enzymatic activity.

The DXO family of proteins participates in eukaryotic mRNA 5'-end quality control, removal of non-canonical NAD+ cap and maturation of fungal rRNA precursors. In this work, we characterize the Arabidopsis thaliana DXO homolog, DXO1. We demonstrate that the plant-specific modification within the active site negatively affects 5'-end capping surveillance properties of DXO1, but has only a minor impact on its strong deNADding activity. Unexpectedly, catalytic activity does not contribute to striking morphological and molecular aberrations observed upon DXO1 knockout in plants, which include growth and pigmentation deficiency, global transcriptomic changes and accumulation of RNA quality control siRNAs. Conversely, these phenotypes depend on the plant-specific N-terminal extension of DXO1. Pale-green coloration of DXO1-deficient plants and our RNA-seq data reveal that DXO1 affects chloroplast-localized processes. We propose that DXO1 mediates the connection between RNA turnover and retrograde chloroplast-to-nucleus signaling independently of its deNADding properties.

Defective XRN3-mediated transcription termination in Arabidopsis affects the expression of protein-coding genes.

Arabidopsis thaliana contains two nuclear XRN2/3 5'-3' exonucleases that are homologs of yeast and human Rat1/Xrn2 proteins involved in the processing and degradation of several classes of nuclear RNAs and in transcription termination of RNA polymerase II. Using strand-specific short read sequencing we show that knockdown of XRN3 leads to an altered expression of hundreds of genes and the accumulation of uncapped and polyadenylated read-through transcripts generated by inefficiently terminated Pol II. Our data support the notion that XRN3-mediated changes in the expression of a subset of genes are caused by upstream read-through transcription and these effects are enhanced by RNA-mRNA chimeras generated in xrn3 plants. In turn, read-through transcripts that are antisense to downstream genes may trigger production of siRNA. Our results highlight the importance of XRN3 exoribonuclease in Pol II transcription termination in plants and show that disturbance in this process may significantly alter gene expression.

Dormancy heterogeneity among Arabidopsis thaliana seeds is linked to individual seed size.

Production of morphologically and physiologically variable seeds is an important strategy that helps plants to survive in unpredictable natural conditions. However, the model plant Arabidopsis thaliana and most agronomically essential crops produce visually homogenous seeds. Using automated phenotype analysis, we observed that small seeds in Arabidopsis tend to have higher primary and secondary dormancy levels than large seeds. Transcriptomic analysis revealed distinct gene expression profiles between large and small seeds. Large seeds have higher expression of translation-related genes implicated in germination competence. By contrast, small seeds have elevated expression of many positive regulators of dormancy, including a key regulator of this process, the DOG1 gene. Differences in DOG1 expression are associated with differential production of its alternative cleavage and polyadenylation isoforms; in small seeds, the proximal poly(A) site is selected, resulting in a short mRNA isoform. Furthermore, single-seed RNA sequencing analysis demonstrated that large seeds resemble DOG1 knockout mutant seeds. Finally, on the single-seed level, expression of genes affected by seed size is correlated with expression of genes that position seeds on the path toward germination. Our results demonstrate an unexpected link between seed size and dormancy phenotypes in a species that produces highly homogenous seed pools, suggesting that the correlation between seed morphology and physiology is more widespread than initially assumed.

The UBP5 histone H2A deubiquitinase counteracts PRCs-mediated repression to regulate Arabidopsis development.

Polycomb Repressive Complexes (PRCs) control gene expression through the incorporation of H2Aub and H3K27me3. In recent years, there is increasing evidence of the complexity of PRCs' interaction networks and the interplay of these interactors with PRCs in epigenome reshaping, which is fundamental to understand gene regulatory mechanisms. Here, we identified UBIQUITIN SPECIFIC PROTEASE 5 (UBP5) as a chromatin player able to counteract the deposition of the two PRCs' epigenetic hallmarks in Arabidopsis thaliana. We demonstrated that UBP5 is a plant developmental regulator based on functional analyses of ubp5-CRISPR Cas9 mutant plants. UBP5 promotes H2A monoubiquitination erasure, leading to transcriptional de-repression. Furthermore, preferential association of UBP5 at PRC2 recruiting motifs and local H3K27me3 gaining in ubp5 mutant plants suggest the existence of functional interplays between UBP5 and PRC2 in regulating epigenome dynamics. In summary, acting as an antagonist of the pivotal epigenetic repressive marks H2Aub and H3K27me3, UBP5 provides novel insights to disentangle the complex regulation of PRCs' activities.

Analysis of mRNA-derived siRNAs in mutants of mRNA maturation and surveillance pathways in Arabidopsis thaliana.

Defects in RNA maturation and RNA decay factors may generate substrates for the RNA interference machinery. This phenomenon was observed in plants where mutations in some RNA-related factors lead to the production of RNA-quality control small interfering RNAs and several mutants show enhanced silencing of reporter transgenes. To assess the potential of RNAi activation on endogenous transcripts, we sequenced small RNAs from a set of Arabidopsis thaliana mutants with defects in various RNA metabolism pathways. We observed a global production of siRNAs caused by inefficient pre-mRNA cleavage and polyadenylation leading to read-through transcription into downstream antisense genes. In addition, in the lsm1a lsm1b double mutant, we identified NIA1, SMXL5, and several miRNA-targeted mRNAs as producing siRNAs, a group of transcripts suggested being especially sensitive to deficiencies in RNA metabolism. However, in most cases, RNA metabolism perturbations do not lead to the widespread production of siRNA derived from mRNA molecules. This observation is contrary to multiple studies based on reporter transgenes and suggests that only a very high accumulation of defective mRNA species caused by specific mutations or substantial RNA processing defects trigger RNAi pathways.

Arabidopsi s Spliceosome Factor SmD3 Modulates Immunity to Pseudomonas syringae Infection.

SmD3 is a core component of the small nuclear ribonucleoprotein (snRNP) that is essential for pre-mRNA splicing. The role of Arabidopsis SmD3 in plant immunity was assessed by testing sensitivity of smd3a and smd3b mutants to Pseudomonas syringae pv. tomato (Pst) DC3000 infection and its pathogenesis effectors flagellin (flg22), EF-Tu (elf18) and coronatine (COR). Both smd3 mutants exhibited enhanced susceptibility to Pst accompanied by marked changes in the expression of key pathogenesis markers. mRNA levels of major biotic stress response factors were also altered upon treatment with Pseudomonas effectors. Our genome-wide transcriptome analysis of the smd3b-1 mutant infected with Pst, verified by northern and RT-qPCR, showed that lack of SmD3-b protein deregulates defense against Pst infection at the transcriptional and posttranscriptional levels including defects in splicing and an altered pattern of alternative splicing. Importantly, we show that SmD3-b dysfunction impairs mainly stomatal immunity as a result of defects in stomatal development. We propose that it is the malfunction of the stomata that is the primary cause of an altered mutant response to the pathogen. Other changes in the smd3b-1 mutant involved enhanced elf18- and flg22-induced callose deposition, reduction of flg22-triggered production of early ROS and boost of secondary ROS caused by Pst infection. Together, our data indicate that SmD3 contributes to the plant immune response possibly via regulation of mRNA splicing of key pathogenesis factors.

Long non-coding RNA produced by RNA polymerase V determines boundaries of heterochromatin.

RNA-mediated transcriptional gene silencing is a conserved process where small RNAs target transposons and other sequences for repression by establishing chromatin modifications. A central element of this process are long non-coding RNAs (lncRNA), which in Arabidopsis thaliana are produced by a specialized RNA polymerase known as Pol V. Here we show that non-coding transcription by Pol V is controlled by preexisting chromatin modifications located within the transcribed regions. Most Pol V transcripts are associated with AGO4 but are not sliced by AGO4. Pol V-dependent DNA methylation is established on both strands of DNA and is tightly restricted to Pol V-transcribed regions. This indicates that chromatin modifications are established in close proximity to Pol V. Finally, Pol V transcription is preferentially enriched on edges of silenced transposable elements, where Pol V transcribes into TEs. We propose that Pol V may play an important role in the determination of heterochromatin boundaries.

Rat1 and Xrn2: The Diverse Functions of the Nuclear Rat1/Xrn2 Exonuclease.

The role of the nucleus of a eukaryotic cell during gene expression is not only limited to transcription and RNA processing but also includes the initial stages of RNA surveillance. All of these processes, and more precisely, transcription elongation and termination, 5'-end RNA maturation, and the removal of processing intermediates and aberrant molecules, require the activity of the nuclear 5'-3' exoribonuclease Rat1/Xrn2. This protein, together with its cytoplasmic counterpart, Xrn1, constitutes a highly conserved eukaryotic family of nucleases, whose roles exceed participation in RNA metabolism alone. Despite many years of extensive research and recent findings related to the structure and function of these enzymes revealed almost every year, several aspects are yet to be discovered.