Optimizing ChIRP-MS for Comprehensive Profiling of RNA-Protein Interactions in Arabidopsis thaliana: A Telomerase RNA Case Study.

The current repertoire of methods available for studying RNA-protein interactions in plants is somewhat limited. Employing an RNA-centric approach, particularly with less abundant RNAs, presents various challenges. Many of the existing methods were initially designed for different model systems, with their application in plants receiving limited attention thus far. The Comprehensive Identification of RNA-Binding Proteins by Mass Spectrometry (ChIRP-MS) technique, initially developed for mammalian cells, has been adapted in this study for application in Arabidopsis thaliana. The procedures have been meticulously modified and optimized for telomerase RNA, a notable example of a low-abundance RNA recently identified. Following these optimization steps, ChIRP-MS can serve as an effective screening method for identifying candidate proteins interacting with any target RNA of interest.

KU70 and CAF-1 in Arabidopsis: Divergent roles in rDNA stability and telomere homeostasis.

Deficiency in chromatin assembly factor-1 (CAF-1) in plants through dysfunction of its components, FASCIATA1 and 2 (FAS1, FAS2), leads to the specific and progressive loss of rDNA and telomere repeats in plants. This loss is attributed to defective repair mechanisms for the increased DNA breaks encountered during replication, a consequence of impaired replication-dependent chromatin assembly. In this study, we explore the role of KU70 in these processes. Our findings reveal that, although the rDNA copy number is reduced in ku70 mutants when compared with wild-type plants, it is not markedly affected by diverse KU70 status in fas1 mutants. This is consistent with our previous characterisation of rDNA loss in fas mutants as a consequence part of the single-strand annealing pathway of homology-dependent repair. In stark contrast to rDNA, KU70 dysfunction fully suppresses the loss of telomeres in fas1 plants and converts telomeres to their elongated and heterogeneous state typical for ku70 plants. We conclude that the alternative telomere lengthening pathway, known to be activated in the absence of KU70, overrides progressive telomere loss due to CAF-1 dysfunction.

In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants.

Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as fasciata1 and nucleolin1 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

Epigenetics and plant hormone dynamics - a functional and methodological perspective.

Plant hormones, pivotal regulators of plant growth, development, and response to environmental cues, have recently emerged as central modulators of epigenetic processes governing gene expression and phenotypic plasticity. This review addresses the complex interplay between plant hormones and epigenetic mechanisms, highlighting the diverse methodologies that have been harnessed to decipher these intricate relationships. We present a comprehensive overview to understand how phytohormones orchestrate epigenetic modifications, shaping plant adaptation and survival strategies. Conversely, we explore how epigenetic regulators ensure hormonal balance and regulate the signalling pathways of key plant hormones. Furthermore, our investigation includes a search for novel genes that are regulated by plant hormones under the control of epigenetic processes. Our review offers a contemporary overview of the epigenetic-plant hormone crosstalk, emphasizing its significance in plant growth, development, and potential agronomical applications.

TeloBase: a community-curated database of telomere sequences across the tree of life.

Discoveries over the recent decade have demonstrated the unexpected diversity of telomere DNA motifs in nature. However, currently available resources, 'Telomerase database' and 'Plant rDNA database', contain just fragments of all relevant literature published over decades of telomere research as they have a different primary focus and limited updates. To fill this gap, we gathered data about telomere DNA sequences from a thorough literature screen as well as by analysing publicly available NGS data, and we created TeloBase (http://cfb.ceitec.muni.cz/telobase/) as a comprehensive database of information about telomere motif diversity. TeloBase is supplemented by internal taxonomy utilizing popular on-line taxonomic resources that enables in-house data filtration and graphical visualisation of telomere DNA evolutionary dynamics in the form of heat tree plots. TeloBase avoids overreliance on administrators for future data updates by having a simple form and community-curation system for application and approval, respectively, of new telomere sequences by users, which should ensure timeliness of the database and topicality. To demonstrate TeloBase utility, we examined telomere motif diversity in species from the fungal genus Aspergillus, and discovered (TTTATTAGGG)n sequence as a putative telomere motif in the plant family Chrysobalanaceae. This was bioinformatically confirmed by analysing template regions of identified telomerase RNAs.

Analysis of Telomerase RNA Structure in Physcomitrium patens Indicates Functionally Relevant Transitions Between OPEN and CLOSED Conformations.

Telomerase RNA (TR) conformation determines its function as a template for telomere synthesis and as a scaffold for the assembly of the telomerase nucleoprotein complex. Experimental analyses of TR secondary structure using DMS-Map Seq and SHAPE-Map Seq techniques show its CLOSED conformation as the consensus structure where the template region cannot perform its function. Our data show that the apparent discrepancy between experimental results and predicted TR functional conformation, mostly ignored in published studies, can be explained using data analysis based on single-molecule structure prediction from individual sequencing reads by the recently established DaVinci method. This method results in several clusters of secondary structures reflecting the structural dynamics of TR, possibly related to its multiple functional states. Interestingly, the presumed active (OPEN) conformation of TR corresponds to a minor fraction of TR under in vivo conditions. Therefore, structural polymorphism and dynamic TR transitions between CLOSED and OPEN conformations may be involved in telomerase activity regulation as a switch that functions independently of total TR transcript levels.

Unraveling Epigenetic Changes in A. thaliana Calli: Impact of HDAC Inhibitors.

The ability for plant regeneration from dedifferentiated cells opens up the possibility for molecular bioengineering to produce crops with desirable traits. Developmental and environmental signals that control cell totipotency are regulated by gene expression via dynamic chromatin remodeling. Using a mass spectrometry-based approach, we investigated epigenetic changes to the histone proteins during callus formation from roots and shoots of Arabidopsis thaliana seedlings. Increased levels of the histone H3.3 variant were found to be the major and most prominent feature of 20-day calli, associated with chromatin relaxation. The methylation status in root- and shoot-derived calli reached the same level during long-term propagation, whereas differences in acetylation levels provided a long-lasting imprint of root and shoot origin. On the other hand, epigenetic signs of origin completely disappeared during 20 days of calli propagation in the presence of histone deacetylase inhibitors (HDACi), sodium butyrate, and trichostatin A. Each HDACi affected the state of post-translational histone modifications in a specific manner; NaB-treated calli were epigenetically more similar to root-derived calli, and TSA-treated calli resembled shoot-derived calli.

Telomerase RNA gene paralogs in plants - the usual pathway to unusual telomeres.

Telomerase, telomeric DNA and associated proteins together represent a complex, finely tuned and functionally conserved mechanism that ensures genome integrity by protecting and maintaining chromosome ends. Changes in its components can threaten an organism's viability. Nevertheless, molecular innovation in telomere maintenance has occurred multiple times during eukaryote evolution, giving rise to species/taxa with unusual telomeric DNA sequences, telomerase components or telomerase-independent telomere maintenance. The central component of telomere maintenance machinery is telomerase RNA (TR) as it templates telomere DNA synthesis, its mutation can change telomere DNA and disrupt its recognition by telomere proteins, thereby leading to collapse of their end-protective and telomerase recruitment functions. Using a combination of bioinformatic and experimental approaches, we examine a plausible scenario of evolutionary changes in TR underlying telomere transitions. We identified plants harbouring multiple TR paralogs whose template regions could support the synthesis of diverse telomeres. In our hypothesis, formation of unusual telomeres is associated with the occurrence of TR paralogs that can accumulate mutations, and through their functional redundancy, allow for the adaptive evolution of the other telomere components. Experimental analyses of telomeres in the examined plants demonstrate evolutionary telomere transitions corresponding to TR paralogs with diverse template regions.

Histone H1 protects telomeric repeats from H3K27me3 invasion in Arabidopsis.

While the pivotal role of linker histone H1 in shaping nucleosome organization is well established, its functional interplays with chromatin factors along the epigenome are just starting to emerge. Here we show that, in Arabidopsis, as in mammals, H1 occupies Polycomb Repressive Complex 2 (PRC2) target genes where it favors chromatin condensation and H3K27me3 deposition. We further show that, contrasting with its conserved function in PRC2 activation at genes, H1 selectively prevents H3K27me3 accumulation at telomeres and large pericentromeric interstitial telomeric repeat (ITR) domains by restricting DNA accessibility to Telomere Repeat Binding (TRB) proteins, a group of H1-related Myb factors mediating PRC2 cis recruitment. This study provides a mechanistic framework by which H1 avoids the formation of gigantic H3K27me3-rich domains at telomeric sequences and contributes to safeguard nucleus architecture.

Visualization of the Nucleolus Using 5' Ethynyl Uridine.

Labeling of the nucleolus in Arabidopsis thaliana can be achieved by incorporation of 5'-ethynyl uridine (EU) into bulk RNA. Although EU does not selectively label the nucleolus, the abundance of ribosomal transcripts results in the predominant accumulation of the signal in the nucleolus. Ethynyl uridine has the advantage of being detected via Click-iT chemistry providing a specific signal and low background. While the protocol presented here employs fluorescent dye and allows visualization of the nucleolus by microscopy, this method can also be used for other downstream applications. Though we tested nucleolar labeling only in A. thaliana, in principle it can be applied to other plant species.

RUVBL proteins are involved in plant gametophyte development.

The proper development of male and female gametophytes is critical for successful sexual reproduction and requires a carefully regulated series of events orchestrated by a suite of various proteins. RUVBL1 and RUVBL2, plant orthologues of human Pontin and Reptin, respectively, belong to the evolutionarily highly conserved AAA+ family linked to a wide range of cellular processes. Previously, we found that RUVBL1 and RUVBL2A mutations are homozygous lethal in Arabidopsis. Here, we report that RUVBL1 and RUVBL2A play roles in reproductive development. We show that mutant plants produce embryo sacs with an abnormal structure or with various numbers of nuclei. Although pollen grains of heterozygous mutant plants exhibit reduced viability and reduced pollen tube growth in vitro, some of the ruvbl pollen tubes are capable of targeting ovules in vivo. Similarly, some ruvbl ovules retain the ability to attract wild-type pollen tubes but fail to develop further. The activity of the RUVBL1 and RUVBL2A promoters was observed in the embryo sac, pollen grains, and tapetum cells and, for RUVBL2A, also in developing ovules. In summary, we show that the RUVBL proteins are essential for the proper development of both male and particularly female gametophytes in Arabidopsis.

Telomerase RNA in Hymenoptera (Insecta) switched to plant/ciliate-like biogenesis.

In contrast to the catalytic subunit of telomerase, its RNA subunit (TR) is highly divergent in size, sequence and biogenesis pathways across eukaryotes. Current views on TR evolution assume a common origin of TRs transcribed with RNA polymerase II in Opisthokonta (the supergroup including Animalia and Fungi) and Trypanosomida on one hand, and TRs transcribed with RNA polymerase III under the control of type 3 promoter, found in TSAR and Archaeplastida supergroups (including e.g. ciliates and Viridiplantae taxa, respectively). Here, we focus on unknown TRs in one of the largest Animalia order - Hymenoptera (Arthropoda) with more than 300 available representative genomes. Using a combination of bioinformatic and experimental approaches, we identify their TRs. In contrast to the presumed type of TRs (H/ACA box snoRNAs transcribed with RNA Polymerase II) corresponding to their phylogenetic position, we find here short TRs of the snRNA type, likely transcribed with RNA polymerase III under the control of the type 3 promoter. The newly described insect TRs thus question the hitherto assumed monophyletic origin of TRs across Animalia and point to an evolutionary switch in TR type and biogenesis that was associated with the divergence of Arthropods.

Sulfate supplementation affects nutrient and photosynthetic status of Arabidopsis thaliana and Nicotiana tabacum differently under prolonged exposure to cadmium.

Hydroponic experiments were performed to examine the effect of prolonged sulfate limitation combined with cadmium (Cd) exposure in Arabidopsis thaliana and a potential Cd hyperaccumulator, Nicotiana tabacum. Low sulfate treatments (20 and 40 µM MgSO4) and Cd stress (4 µM CdCl2) showed adverse effects on morphology, photosynthetic and biochemical parameters and the nutritional status of both species. For example, Cd stress decreased NO3- root content under 20 µM MgSO4 to approximately 50% compared with respective controls. Interestingly, changes in many measured parameters, such as chlorophyll and carotenoid contents, the concentrations of anions, nutrients and Cd, induced by low sulfate supply, Cd exposure or a combination of both factors, were species-specific. Our data showed opposing effects of Cd exposure on Ca, Fe, Mn, Cu and Zn levels in roots of the studied plants. In A. thaliana, levels of glutathione, phytochelatins and glucosinolates demonstrated their distinct involvement in response to sub-optimal growth conditions and Cd stress. In shoot, the levels of phytochelatins and glucosinolates in the organic sulfur fraction were not dependent on sulfate supply under Cd stress. Altogether, our data showed both common and species-specific features of the complex plant response to prolonged sulfate deprivation and/or Cd exposure.

Compromised function of ARM, the interactor of Arabidopsis telomerase, suggests its role in stress responses.

ARM was identified previously as an interaction partner of the telomerase protein subunit (TERT) in Arabidopsis thaliana. To investigate the interconnection between ARM and telomerase and to identify ARM cellular functions, we analyzed a set of arm mutant lines and arm/tert double mutants. Telomere length was not affected in arm single mutant plants, in contrast to double mutants. In the second generation of homozygous arm-1/tert double mutants following the heterozygous state during the double mutant construction, telomeres shortened dramatically, even below levels in tert plants displaying severe morphological defects. Intriguingly, homozygous arm-1/tert double mutants with short telomeres grew without obvious phenotypic changes for next two generations. Then, in agreement with the onset of phenotypic changes in tert, morphological defects were timed to the 5th arm-1/tert homozygous generation. RNAseq analyses of arm-1/tert and respective single mutants displayed markedly overlapping sets of differentially expressed genes in arm-1/tert double mutant and arm-1 single mutant lines, indicating a dominant effect of the ARM mutation. RNAseq data further implied ARM involvement in circadian rhythms, responses to drugs and to biotic and abiotic stimuli. In agreement with it, we observed sensitivity of arm-1 single mutant to the heat stress during germination. Altogether, our results suggest ARM involvement in crucial cellular processes without evidencing its role in the telomerase canonical function.

MEDIATOR SUBUNIT17 integrates jasmonate and auxin signaling pathways to regulate thermomorphogenesis.

Plant adjustment to environmental changes involves complex crosstalk between extrinsic and intrinsic cues. In the past two decades, extensive research has elucidated the key roles of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and the phytohormone auxin in thermomorphogenesis. In this study, we identified a previously unexplored role of jasmonate (JA) signaling components, the Mediator complex, and their integration with auxin signaling during thermomorphogenesis in Arabidopsis (Arabidopsis thaliana). Warm temperature induces expression of JA signaling genes including MYC2, but, surprisingly, this transcriptional activation is not JA dependent. Warm temperature also promotes accumulation of the JA signaling receptor CORONATINE INSENSITIVE1 (COI1) and degradation of the JA signaling repressor JASMONATE-ZIM-DOMAIN PROTEIN9, which probably leads to de-repression of MYC2, enabling it to contribute to the expression of MEDIATOR SUBUNIT17 (MED17). In response to warm temperature, MED17 occupies the promoters of thermosensory genes including PIF4, YUCCA8 (YUC8), INDOLE-3-ACETIC ACID INDUCIBLE19 (IAA19), and IAA29. Moreover, MED17 facilitates enrichment of H3K4me3 on the promoters of PIF4, YUC8, IAA19, and IAA29 genes. Interestingly, both occupancy of MED17 and enrichment of H3K4me3 on these thermomorphogenesis-related promoters are dependent on PIF4 (or PIFs). Altered accumulation of COI1 under warm temperature in the med17 mutant suggests the possibility of a feedback mechanism. Overall, this study reveals the role of the Mediator complex as an integrator of JA and auxin signaling pathways during thermomorphogenesis.

GERONIMO: A tool for systematic retrieval of structural RNAs in a broad evolutionary context.

BACKGROUND: While web-based tools such as BLAST have made identifying conserved gene homologs appear easy, genes with variable sequences pose significant challenges. Functionally important noncoding RNAs (ncRNA) often show low sequence conservation due to genetic variations, including insertions and deletions. Rather than conserved sequences, these RNAs possess highly conserved structural features across a broad phylogenetic range. Such features can be identified using the covariance models approach, which combines sequence alignment with a secondary RNA structure consensus. However, running standard implementation of that approach (Infernal) requires advanced bioinformatics knowledge compared to user-friendly web services like BLAST. The issue is partially addressed by RNAcentral, which can be used to search for homologs across a broad range of ncRNA sequence collections from diverse organisms but not across the genome assemblies. RESULTS: Here, we present GERONIMO, which conducts evolutionary searches across hundreds of genomes in a fully automated way. It provides results extended with taxonomy context, as summary tables and visualizations, to facilitate analysis for user convenience. Additionally, GERONIMO supplements homologous sequences with genomic regions to analyze promoter motifs or gene collinearity, enhancing the validation of results. CONCLUSION: GERONIMO, built using Snakemake, has undergone extensive testing on hundreds of genomes, establishing itself as a valuable tool in the identification of ncRNA homologs across diverse taxonomic groups. Consequently, GERONIMO facilitates the investigation of the evolutionary patterns of functionally significant ncRNA players, whose understanding has previously been limited to individual organisms and close relatives.

G2/M-checkpoint activation in fasciata1 rescues an aberrant S-phase checkpoint but causes genome instability.

The WEE1 and ATM AND RAD3-RELATED (ATR) kinases are important regulators of the plant intra-S-phase checkpoint; consequently, WEE1KO and ATRKO roots are hypersensitive to replication-inhibitory drugs. Here, we report on a loss-of-function mutant allele of the FASCIATA1 (FAS1) subunit of the chromatin assembly factor 1 (CAF-1) complex that suppresses the phenotype of WEE1- or ATR-deficient Arabidopsis (Arabidopsis thaliana) plants. We demonstrate that lack of FAS1 activity results in the activation of an ATAXIA TELANGIECTASIA MUTATED (ATM)- and SUPPRESSOR OF GAMMA-RESPONSE 1 (SOG1)-mediated G2/M-arrest that renders the ATR and WEE1 checkpoint regulators redundant. This ATM activation accounts for the telomere erosion and loss of ribosomal DNA that are described for fas1 plants. Knocking out SOG1 in the fas1 wee1 background restores replication stress sensitivity, demonstrating that SOG1 is an important secondary checkpoint regulator in plants that fail to activate the intra-S-phase checkpoint.

Super-resolution microscopy of chromatin fibers and quantitative DNA methylation analysis of DNA fiber preparations.

Analysis of histone variants and epigenetic marks is dominated by genome-wide approaches in the form of chromatin immunoprecipitation-sequencing (ChIP-seq) and related methods. Although uncontested in their value for single-copy genes, mapping the chromatin of DNA repeats is problematic for biochemical techniques that involve averaging of cell populations or analysis of clusters of tandem repeats in a single-cell analysis. Extending chromatin and DNA fibers allows us to study the epigenetics of individual repeats in their specific chromosomal context, and thus constitutes an important tool for gaining a complete understanding of the epigenetic organization of genomes. We report that using an optimized fiber extension protocol is essential in order to obtain more reproducible data and to minimize the clustering of fibers. We also demonstrate that the use of super-resolution microscopy is important for reliable evaluation of the distribution of histone modifications on individual fibers. Furthermore, we introduce a custom script for the analysis of methylation levels on DNA fibers and apply it to map the methylation of telomeres, ribosomal genes and centromeres.

Expansion of rDNA and pericentromere satellite repeats in the genomes of bank voles Myodes glareolus exposed to environmental radionuclides.

Altered copy number of certain highly repetitive regions of the genome, such as satellite DNA within heterochromatin and ribosomal RNA loci (rDNA), is hypothesized to help safeguard the genome against damage derived from external stressors. We quantified copy number of the 18S rDNA and a pericentromeric satellite DNA (Msat-160) in bank voles (Myodes glareolus) inhabiting the Chernobyl Exclusion Zone (CEZ), an area that is contaminated by radionuclides and where organisms are exposed to elevated levels of ionizing radiation. We found a significant increase in 18S rDNA and Msat-160 content in the genomes of bank voles from contaminated locations within the CEZ compared with animals from uncontaminated locations. Moreover, 18S rDNA and Msat-160 copy number were positively correlated in the genomes of bank voles from uncontaminated, but not in the genomes of animals inhabiting contaminated, areas. These results show the capacity for local-scale geographic variation in genome architecture and are consistent with the genomic safeguard hypothesis. Disruption of cellular processes related to genomic stability appears to be a hallmark effect in bank voles inhabiting areas contaminated by radionuclides.

Extraordinary diversity of telomeres, telomerase RNAs and their template regions in Saccharomycetaceae.

Telomerase RNA (TR) carries the template for synthesis of telomere DNA and provides a scaffold for telomerase assembly. Fungal TRs are long and have been compared to higher eukaryotes, where they show considerable diversity within phylogenetically close groups. TRs of several Saccharomycetaceae were recently identified, however, many of these remained uncharacterised in the template region. Here we show that this is mainly due to high variability in telomere sequence. We predicted the telomere sequences using Tandem Repeats Finder and then we identified corresponding putative template regions in TR candidates. Remarkably long telomere units and the corresponding putative TRs were found in Tetrapisispora species. Notably, variable lengths of the annealing sequence of the template region (1-10 nt) were found. Consequently, species with the same telomere sequence may not harbour identical TR templates. Thus, TR sequence alone can be used to predict a template region and telomere sequence, but not to determine these exactly. A conserved feature of telomere sequences, tracts of adjacent Gs, led us to test the propensity of individual telomere sequences to form G4. The results show highly diverse values of G4-propensity, indicating the lack of ubiquitous conservation of this feature across Saccharomycetaceae.