skip to main content

Title: 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.  more » « less
Award ID(s):
2023310 2021753 1758532
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ;
Date Published:
Journal Name:
Nucleic Acids Research
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    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.

    more » « less
  2. null (Ed.)
    Telomerase is essential for maintaining telomere integrity. Although telomerase function is widely conserved, the integral telomerase RNA (TR) that provides a template for telomeric DNA synthesis has diverged dramatically. Nevertheless, TR molecules retain 2 highly conserved structural domains critical for catalysis: a template-proximal pseudoknot (PK) structure and a downstream stem-loop structure. Here we introduce the authentic TR from the plant Arabidopsis thaliana , called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. This RNA is distinct from the RNA previously described as the templating telomerase RNA, AtTER1. AtTR is a 268-nt Pol III transcript necessary for telomere maintenance in vivo and sufficient with TERT to reconstitute telomerase activity in vitro. Bioinformatics analysis identified 85 AtTR orthologs from 3 major clades of plants: angiosperms, gymnosperms, and lycophytes. Through phylogenetic comparisons, a secondary structure model conserved among plant TRs was inferred and verified using in vitro and in vivo chemical probing. The conserved plant TR structure contains a template-PK core domain enclosed by a P1 stem and a 3′ long-stem P4/5/6, both of which resemble a corresponding structural element in ciliate and vertebrate TRs. However, the plant TR contains additional stems and linkers within the template-PK core, allowing for expansion of PK structure from the simple PK in the smaller ciliate TR during evolution. Thus, the plant TR provides an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates. 
    more » « less
  3. Wittkopp, Patricia (Ed.)
    Abstract Telomerase RNA (TR) is a noncoding RNA essential for the function of telomerase ribonucleoprotein. TRs from vertebrates, fungi, ciliates, and plants exhibit extreme diversity in size, sequence, secondary structure, and biogenesis pathway. However, the evolutionary pathways leading to such unusual diversity among eukaryotic kingdoms remain elusive. Within the metazoan kingdom, the study of TR has been limited to vertebrates and echinoderms. To understand the origin and evolution of TR across the animal kingdom, we employed a phylogeny-guided, structure-based bioinformatics approach to identify 82 novel TRs from eight previously unexplored metazoan phyla, including the basal-branching sponges. Synthetic TRs from two representative species, a hemichordate and a mollusk, reconstitute active telomerase in vitro with their corresponding telomerase reverse transcriptase components, confirming that they are authentic TRs. Comparative analysis shows that three functional domains, template-pseudoknot (T-PK), CR4/5, and box H/ACA, are conserved between vertebrate and the basal metazoan lineages, indicating a monophyletic origin of the animal TRs with a snoRNA-related biogenesis mechanism. Nonetheless, TRs along separate animal lineages evolved with divergent structural elements in the T-PK and CR4/5 domains. For example, TRs from echinoderms and protostomes lack the canonical CR4/5 and have independently evolved functionally equivalent domains with different secondary structures. In the T-PK domain, a P1.1 stem common in most metazoan clades defines the template boundary, which is replaced by a P1-defined boundary in vertebrates. This study provides unprecedented insight into the divergent evolution of detailed TR secondary structures across broad metazoan lineages, revealing ancestral and later-diversified elements. 
    more » « less
  4. Abstract

    Promoters regulate both the amplitude and pattern of gene expression—key factors needed for optimization of many synthetic biology applications. Previous work in Arabidopsis found that promoters that contain a TATA-box element tend to be expressed only under specific conditions or in particular tissues, while promoters that lack any known promoter elements, thus designated as Coreless, tend to be expressed more uniformly. To test whether this trend represents a conserved promoter design rule, we identified stably expressed genes across multiple angiosperm species using publicly available RNA-seq data. Comparisons between core promoter architectures and gene expression stability revealed differences in core promoter usage in monocots and eudicots. Furthermore, when tracing the evolution of a given promoter across species, we found that core promoter type was not a strong predictor of expression pattern. Our analysis suggests that core promoter types are correlative rather than causative in promoter expression patterns and highlights the challenges in finding or building constitutive promoters that will work across diverse plant species.

    more » « less
  5. null (Ed.)
    We report the biological and structural characterization of umbravirus-like associated RNAs (ulaRNAs), a new category of coat-protein dependent subviral RNA replicons that infect plants. These RNAs encode an RNA-dependent RNA polymerase (RdRp) following a −1 ribosomal frameshift event, are 2.7–4.6 kb in length, and are related to umbraviruses, unlike similar RNA replicons that are related to tombusviruses. Three classes of ulaRNAs are proposed, with citrus yellow vein associated virus (CYVaV) placed in Class 2. With the exception of CYVaV, Class 2 and Class 3 ulaRNAs encode an additional open reading frame (ORF) with movement protein-like motifs made possible by additional sequences just past the RdRp termination codon. The full-length secondary structure of CYVaV was determined using Selective 2’ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) structure probing and phylogenic comparisons, which was used as a template for determining the putative structures of the other Class 2 ulaRNAs, revealing a number of distinctive structural features. The ribosome recoding sites of nearly all ulaRNAs, which differ significantly from those of umbraviruses, may exist in two conformations and are highly efficient. The 3′ regions of Class 2 and Class 3 ulaRNAs have structural elements similar to those of nearly all umbraviruses, and all Class 2 ulaRNAs have a unique, conserved 3′ cap-independent translation enhancer. CYVaV replicates independently in protoplasts, demonstrating that the reported sequence is full-length. Additionally, CYVaV contains a sequence in its 3′ UTR that confers protection to nonsense mediated decay (NMD), thus likely obviating the need for umbravirus ORF3, a known suppressor of NMD. This initial characterization lays down a road map for future investigations into these novel virus-like RNAs. 
    more » « less