Evolutionary and biochemical analyses reveal conservation of the Brassicaceae telomerase ribonucleoprotein complex

Abstract

<jats:title>Abstract</jats:title><jats:p>The telomerase ribonucleoprotein complex (RNP) is essential for genome stability and performs this role through the addition of repetitive DNA to the ends of chromosomes. The telomerase enzyme is composed of a reverse transcriptase (TERT), which utilizes a template domain in an RNA subunit (TER) to reiteratively add telomeric DNA at the ends of chromosomes. Multiple TERs have been identified in the model plant <jats:italic>Arabidopsis thaliana</jats:italic>. Here we combine a phylogenetic and biochemical approach to understand how the telomerase RNP has evolved in Brassicaceae, the family that includes <jats:italic>A. thaliana</jats:italic>. Because of the complex phylogenetic pattern of template domain loss and alteration at the previously characterized <jats:italic>A. thaliana</jats:italic> TER loci, <jats:italic>TER1</jats:italic> and <jats:italic>TER2</jats:italic>, across the plant family Brassicaceae, we bred double mutants from plants with a template deletion at <jats:italic>AtTER1</jats:italic> and T-DNA insertion at <jats:italic>AtTER2</jats:italic>. These double mutants exhibited no telomere length deficiency, a definitive indication that neither of these loci encode a functional telomerase RNA. Moreover, we determined that the telomerase components <jats:italic>TERT, Dyskerin</jats:italic>, and the <jats:italic>KU</jats:italic> heterodimer are under strong purifying selection, consistent with the idea that the TER with which they interact is also conserved. To test this hypothesis further, we analyzed the substrate specificity of telomerase from species across Brassicaceae and determined that telomerase from close relatives bind and extend substrates in a similar manner, supporting the idea that TERs in different species are highly similar to one another and are likely encoded from an orthologous locus. Lastly, TERT proteins from across Brassicaceae were able to complement loss of function <jats:italic>tert</jats:italic> mutants <jats:italic>in vivo</jats:italic>, indicating TERTs from other species have the ability to recognize the native TER of <jats:italic>A. thaliana</jats:italic>. Finally, we immunoprecipitated the telomerase complex and identified associated RNAs via RNA-seq. Using our evolutionary data we constrained our analyses to conserved RNAs within Brassicaceae that contained a template domain. These analyses revealed a highly expressed locus whose disruption by a T-DNA resulted in a telomeric phenotype similar to the loss of other telomerase core proteins, indicating that the RNA has an important function in telomere maintenance.</jats:p>

The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans

“…S1 B and C). These results are inconsistent with AtTER1 being a functional telomerase RNA and instead support the recent findings of Fajkus et al (34) and Dew-Budd et al (35) indicating that AtTER1 is not required for telomere maintenance.…”

“…We previously described the identification of 2 telomeraseassociated RNAs from Arabidopsis thaliana, termed AtTER1 and AtTER2 (32,33). AtTER1 was proposed to serve as the template for telomeric DNA synthesis by telomerase (32); however, recent data have refuted the role of AtTER1 in telomere maintenance (34,35). Moreover, Fajkus et al (34) recently reported the identification of a novel telomerase RNA from A. thaliana, termed AtTR, that is required for telomere maintenance and is conserved across land plants.…”

Origin, Diversity, and Evolution of Telomere Sequences in Plants

“…As far as we know, there is still no data on any algal TR, which would elucidate whether Pol III transcription of TR is a general feature for all plants or not. This work (Fajkus et al, 2019), based on CRISPR knock-out and other experiments, also showed that a previously identified telomerase RNA candidate in A. thaliana (Cifuentes-Rojas et al, 2011;Beilstein et al, 2012) was not a functional template subunit of telomerase, as was also demonstrated shortly after by (Dew-Budd et al, 2019). Assuming that the Pol II/Pol III dependency for TR transcription is a reliable evolutionary marker, future TR research in other main eukaryotic lineages will probably open new insights into the origin of eukaryotes.…”

Human-like telomeres in Zostera marina reveal a mode of transition from the plant to the human telomeric sequences

“…The assembly was performed as stranded RNA-seq with paired-end fastq datasets. Putative TR from Z. marina were found out using blast with published orthologs 10,18,19 .…”

“…Moreover, a combination of the available genomic data from Alismatales with the recent breakthrough of the discovery of plant telomerase RNA10,18,19 have led us to the astonishing conclusion that the template region in plant telomerase RNA is a very dynamic element, with unexpectedly variable borders, even in species with the same telomere sequence. The finding of two distinct telomerase RNA paralogs within several of the Alismatales genomes analysed, including the Z. marina genome assembly, offers an answer to how plants are able to change their telomeres.…”