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

Abstract

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.

Graphical Abstract

We present a smart strategy of telomerase RNA (TR) identification based on its conserved type-3 RNA Pol III promoter and TR template elements. We characterize TRs in early diverging Viridiplantae taxa, as well as in ciliates and other Diaphoretickes lineages. TRs are validated experimentally and show conservation of core TR structural domains. These results shed light on the evolution of a key eukaryotic non-coding RNA across more than a billion years.

Figure 1.

Cladogram of the eukaryote megagroup Diaphoretickes (according to (15)). Taxa with a TR gene identified in this study are highlited by a red asterisk, taxa with a known TR sequence by a black asterisk. These include OIigohymenophorea and Spirotrichea from Ciliates on the one side, and Tracheophytes on the other side. TRs in both, substantially distant, groups share the type 3 RNAPIII promoter which is characterized by its relatively conserved promoter sequence motif termed as Upstream Sequence Element (USE). In this study, we tested a hypothesis that TRs in other clades from Diaphoretickes also have the type 3 promoter whose USE sequence could be exploited for filtering/prediction of novel TR candidates.

Figure 2.

A comprehensive diagram showing key methodological aspects of this study. These include: (A) schematic view of a type 3 promoter including typical type 3 promoter-driven snRNA genes; (B) TR characterization strategy based on our assumption that TR promoter type can be far more conserved through evolution than TR sequence; (C) a detailed workflow of TR identification starting with USE characterization (in purple), followed by TR candidate prediction based on conserved USE and putative template sequence (in yellow), subsequent homology searches (in green) and finally experimental validation of novel TRs (in blue).

Figure 3.

Graphical alignment showing TR identification by homology searches in related genomes. De novo predicted TR candidate (based on USE and putative TR template) from A. protothecoides showed homologs in close relatives P. cutis and P. wickerhamii by using BLASTn. Their putative transcribed regions were used to generate a Covariance model (CM) for searching TR homologs in evolutionarily more distant species by the Infernal tool. New significant hits (e-value ≤ 1e–9) were manually checked for corresponding template, USE or other conserved regions, and used for optimization of CM for reiterative Infernal searches.

Figure 4.

Summary of TR Infernal searches across Viridiplantae illustrated by Venn diagram confirms a homology of newly predicted TRs in this study with previously published TRs in Tracheophytes (5). Three phylogenetically discrete CMs (circles distinguished by colour) were optimized from previously published TRs from Tracheophytes (T, in green), bryophytes and streptophyte algae TRs (BSA, in yellow) and green algae TRs (GA, in blue), respectively (Supplementary File S2). Infernal search with these models (T; BSA; GA) against Viridiplantae TRs (Supplementary Table S4) identified concurrently corresponding TR sequences—visualized as numbers (for respective Venn diagram subsets) distinguished by colour (Tracheophyta TRs—green, bryophytes and streptophyte algae TRs—yellow, green algae TRs—blue).

Figure 5.

Evidence for the presence of transcripts of example predicted TRs from Viridiplantae employing: (A) TR presence in total RNA-seq data from rRNA depleted libraries.TR lengths were estimated based on the lengths of mapped RNA-seq data to reference genomes (if available) or de-novo assembled transcripts; (B) Northern hybridization using corresponding radioactively labelled TR probe (NB). Gels were stained with SYBR™ Gold and visualized in UV light (UV lanes). Low Range ssRNA Ladder (NEB) was used as a marker (M); (C) RT-PCR with TR specific primers (Supplementary File S1). GeneRuler 100 bp DNA Ladder (Thermo Scientific) was used as a marker (M).

Figure 6.

A comparison of type 3 promoters of TRs with other snRNAs that are transcribed with either RNAPII or RNAPIII. A similarity with promoters of respective snRNAs is indicated by ‘∼‘. Mutual position of USE and TATA box (or TATA presence) is crucial for RNAP specificity (12,13,44). USE and TATA box (if present) are in capital letters in alignments. All analysed TR promoters correspond to snRNAs transcribed predominantly by RNAPIII (i.e. U6, SRP snRNA across eukaryotes, or U3 snRNA in Viridiplantae (46,47)).

Figure 7.

Species with diverse telomere DNA motifs (on the left, in green) and their putative TR template regions (on the right). 1 – previously published telomere motifs, 2 – newly identified telomere motifs in this work (Supplementary Table S3). Putative template regions are in capital letters, consisting of a template sequence (in red) and an annealing sequence (in blue). An example of telomerase RNA annealing to the 3′ end of telomere DNA is shown in a bottom part.

Figure 8.

Analysis of P. patens TR knock-out lines (pptr) shows telomere shortening and the loss of telomerase activity. (A) Terminal restriction fragment (TRF) analysis of regenerated P. patens pptr lines (samples 3, 4, 5) and wild-type plants (WT) (samples 1, 2) used as a background for TR knock-outs. For clarity, TRF signals under different exposition intensity are enclosed alongside. (B) Evaluation of telomere lengths from TRF signals using WALTER toolset v2.0. (C) Complete and partial DNA digestions of DNA of WT and pptr line (sample 3 in panel A) with decreasing activities of Tru1I (as indicated above lanes). While partial digestion of WT sample results in a shift of a smeared pattern of TRFs towards higher molecular weight, pptr sample shows a ladder of monomers, dimers and longer arrays of major and minor products (denoted by filled and empty arrowheads, respectively), Product lengths were evaluated using Clinx Image Analysis software (Clinx Science Instruments). (D) TRAP assay using cell extracts (250 ng of total protein) of WT (samples 1, 2) and pptr plants (samples 3, 4). Negative control (nc) indicates reaction without extract.

Figure 9.

Comparative phylogenetic structural predictions of Viridiplantae TRs reveal a deeply conserved core domain. (A) Predicted core Template/Pseudoknot (TPK) region of Bryophyta TRs based on the Physcomitrium patens TR. Invariant sites across the multiple sequence alignment (MSA) of 11 putative TRs are shown in red, with sites >90% conserved shown in purple and covariant sites where, for example, G = C binding switched to A = U binding, are shown in blue. (B) The predicted TPK region for Chlamydomonadales based off of a MSA of seven putative TR loci with Chlamydomonadales reinhardtii as reference. (C) Predicted TPK region for Marchantiophyta based off of a MSA of 12 putative TRs, with Marchantia polyporpha used as reference. (D) A proposed structural model of Viridiplantae TRs based off of the consensus sequences from Bryophyta, Chlamydomonadales, Marchantiophyta and Tracheophytes TPK regions. Full predicted structures or sequence alignments used to build structures are available in Supplementary Figures S2 and S3.