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.

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.

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.

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.

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.

Evolution of plant telomerase RNAs: farther to the past, deeper to the roots.

The enormous sequence heterogeneity of telomerase RNA (TR) subunits has thus far complicated their characterization in a wider phylogenetic range. Our recent finding that land plant TRs are, similarly to known ciliate TRs, transcribed by RNA polymerase III and under the control of the type-3 promoter, allowed us to design a novel strategy to characterize TRs in early diverging Viridiplantae taxa, as well as in ciliates and other Diaphoretickes lineages. Starting with the characterization of the upstream sequence element of the type 3 promoter that is conserved in a number of small nuclear RNAs, and the expected minimum TR template region as search features, we identified candidate TRs in selected Diaphoretickes genomes. Homologous TRs were then used to build covariance models to identify TRs in more distant species. Transcripts of the identified TRs were confirmed by transcriptomic data, RT-PCR and Northern hybridization. A templating role for one of our candidates was validated in Physcomitrium patens. Analysis of secondary structure demonstrated a deep conservation of motifs (pseudoknot and template boundary element) observed in all published TRs. These results elucidate the evolution of the earliest eukaryotic TRs, linking the common origin of TRs across Diaphoretickes, and underlying evolutionary transitions in telomere repeats.

WALTER: an easy way to online evaluate telomere lengths from terminal restriction fragment analysis.

BACKGROUND: Telomeres, nucleoprotein structures comprising short tandem repeats and delimiting the ends of linear eukaryotic chromosomes, play an important role in the maintenance of genome stability. Therefore, the determination of the length of telomeres is of high importance for many studies. Over the last years, new methods for the analysis of the length of telomeres have been developed, including those based on PCR or analysis of NGS data. Despite that, terminal restriction fragment (TRF) method remains the gold standard to this day. However, this method lacks universally accepted and precise tool capable to analyse and statistically evaluate TRF results. RESULTS: To standardize the processing of TRF results, we have developed WALTER, an online toolset allowing rapid, reproducible, and user-friendly analysis including statistical evaluation of the data. Given its web-based nature, it provides an easily accessible way to analyse TRF data without any need to install additional software. CONCLUSIONS: WALTER represents a major upgrade from currently available tools for the image processing of TRF scans. This toolset enables a rapid, highly reproducible, and user-friendly evaluation of almost any TRF scan including in-house statistical evaluation of the data. WALTER platform together with user manual describing the evaluation of TRF scans in detail and presenting tips and troubleshooting, as well as test data to demo the software are available at https://www.ceitec.eu/chromatin-molecular-complexes-jiri-fajkus/rg51/tab?tabId=125#WALTER and the source code at https://github.com/mlyc93/WALTER .

Telomerase RNAs in land plants.

To elucidate the molecular nature of evolutionary changes of telomeres in the plant order Asparagales, we aimed to characterize telomerase RNA subunits (TRs) in these plants. The unusually long telomere repeat unit in Allium plants (12 nt) allowed us to identify TRs in transcriptomic data of representative species of the Allium genus. Orthologous TRs were then identified in Asparagales plants harbouring telomere DNA composed of TTAGGG (human type) or TTTAGGG (Arabidopsis-type) repeats. Further, we identified TRs across the land plant phylogeny, including common model plants, crop plants, and plants with unusual telomeres. Several lines of functional testing demonstrate the templating telomerase function of the identified TRs and disprove a functionality of the only previously reported plant telomerase RNA in Arabidopsis thaliana. Importantly, our results change the existing paradigm in plant telomere biology which has been based on the existence of a relatively conserved telomerase reverse transcriptase subunit (TERT) associating with highly divergent TRs even between closely related plant taxa. The finding of a monophyletic origin of genuine TRs across land plants opens the possibility to identify TRs directly in transcriptomic or genomic data and/or predict telomere sequences synthesized according to the respective TR template region.