skip to main content

Title: The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans
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 more » 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. « less
Authors:
; ; ; ; ; ; ; ; ;
Award ID(s):
1616078
Publication Date:
NSF-PAR ID:
10204013
Journal Name:
Proceedings of the National Academy of Sciences
Volume:
116
Issue:
49
Page Range or eLocation-ID:
24542 to 24550
ISSN:
0027-8424
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract The recent discovery of the bona-fide telomerase RNA (TR) from plants reveals conserved and unique secondary structure elements and the opportunity for new insight into the telomerase RNP. Here we examine how two highly conserved proteins previously implicated in Arabidopsis telomere maintenance, AtPOT1a and AtNAP57 (dyskerin), engage plant telomerase. We report that AtPOT1a associates with Arabidopsis telomerase via interaction with TERT. While loss of AtPOT1a does not impact AtTR stability, the templating domain is more accessible in pot1a mutants, supporting the conclusion that AtPOT1a stimulates telomerase activity but does not facilitate telomerase RNP assembly. We also show, that despite the absence of a canonical H/ACA binding motif within AtTR, dyskerin binds AtTR with high affinity and specificity in vitro via a plant specific three-way junction (TWJ). A core element of the TWJ is the P1a stem, which unites the 5′ and 3′ ends of AtTR. P1a is required for dyskerin-mediated stimulation of telomerase repeat addition processivity in vitro, and for AtTR accumulation and telomerase activity in vivo. The deployment of vertebrate-like accessory proteins and unique RNA structural elements by Arabidopsis telomerase provides a new platform for exploring telomerase biogenesis and evolution.
  2. 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. Inmore »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.« less
  3. 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 telomeremore »repeats.« less
  4. Telomerase is a ribonucleoprotein complex that counteracts the shortening of chromosome ends due to incomplete replication. Telomerase contains a catalytic core of telomerase reverse transcriptase (TERT) and telomerase RNA (TER). However, what defines TERT and separates it from other reverse transcriptases remains a subject of debate. A recent cryoelectron microscopy map of Tetrahymena telomerase revealed the structure of a previously uncharacterized TERT domain (TRAP) with unanticipated interactions with the telomerase essential N-terminal (TEN) domain and roles in telomerase activity. Both TEN and TRAP are absent in the putative Tribolium TERT that has been used as a model for telomerase for over a decade. To investigate the conservation of TRAP and TEN across species, we performed multiple sequence alignments and statistical coupling analysis on all identified TERTs and find that TEN and TRAP have coevolved as telomerase-specific domains. Integrating the data from bioinformatic analysis and the structure of Tetrahymena telomerase, we built a pseudoatomic model of human telomerase catalytic core that accounts for almost all of the cryoelectron microscopy density in a published map, including TRAP in previously unassigned density as well as telomerase RNA domains essential for activity. This more complete model of the human telomerase catalytic core illustrates howmore »domains of TER and TERT, including the TEN–TRAP complex, can interact in a conserved manner to regulate telomere synthesis.« less
  5. Dutch, Rebecca Ellis. (Ed.)
    ABSTRACT Opium poppy mosaic virus (OPMV) is a recently discovered umbravirus in the family Tombusviridae . OPMV has a plus-sense genomic RNA (gRNA) of 4,241 nucleotides (nt) from which replication protein p35 and p35 extension product p98, the RNA-dependent RNA polymerase (RdRp), are expressed. Movement proteins p27 (long distance) and p28 (cell to cell) are expressed from a 1,440-nt subgenomic RNA (sgRNA2). A highly conserved structure was identified just upstream from the sgRNA2 transcription start site in all umbraviruses, which includes a carmovirus consensus sequence, denoting generation by an RdRp-mediated mechanism. OPMV also has a second sgRNA of 1,554 nt (sgRNA1) that starts just downstream of a canonical exoribonuclease-resistant sequence (xrRNA D ). sgRNA1 codes for a 30-kDa protein in vitro that is in frame with p28 and cannot be synthesized in other umbraviruses. Eliminating sgRNA1 or truncating the p30 open reading frame (ORF) without affecting p28 substantially reduced accumulation of OPMV gRNA, suggesting a functional role for the protein. The 652-nt 3′ untranslated region of OPMV contains two 3′ cap-independent translation enhancers (3′ CITEs), a T-shaped structure (TSS) near its 3′ end, and a Barley yellow dwarf virus -like translation element (BTE) in the central region. Only the BTE is functionalmore »in luciferase reporter constructs containing gRNA or sgRNA2 5′ sequences in vivo , which differs from how umbravirus 3′ CITEs were used in a previous study. Similarly to most 3′ CITEs, the OPMV BTE links to the 5′ end via a long-distance RNA-RNA interaction. Analysis of 14 BTEs revealed additional conserved sequences and structural features beyond the previously identified 17-nt conserved sequence. IMPORTANCE Opium poppy mosaic virus (OPMV) is an umbravirus in the family Tombusviridae . We determined that OPMV accumulates two similarly sized subgenomic RNAs (sgRNAs), with the smaller known to code for proteins expressed from overlapping open reading frames. The slightly larger sgRNA1 has a 5′ end just upstream from a previously predicted xrRNA D site, identifying this sgRNA as an unusually long product produced by exoribonuclease trimming. Although four umbraviruses have similar predicted xrRNA D sites, only sgRNA1 of OPMV can code for a protein that is an extension product of umbravirus ORF4. Inability to generate the sgRNA or translate this protein was associated with reduced gRNA accumulation in vivo . We also characterized the OPMV BTE structure, a 3′ cap-independent translation enhancer (3′ CITE). Comparisons of 13 BTEs with the OPMV BTE revealed additional stretches of sequence similarity beyond the 17-nt signature sequence, as well as conserved structural features not previously recognized in these 3′ CITEs.« less