AtTR Is the Predominant RNA Associated with Active Telomerase in Arabidopsis.

Prompted by collaborative work with the Beilstein laboratory, which indicated that AtTER1 was not the authentic TR component for A. thaliana telomerase (35), we developed an unbiased approach to identify ncRNAs associated with the AtTERT protein through RNA immunoprecipitation (RIP) analysis using anti-AtTERT antibody. RIP was performed under native conditions with mild salt and detergent concentrations to retain weak interactions. Next-generation sequencing of copurified RNAs identified 177 RNA sequences that were significantly enriched in the wild-type (WT) samples but not in the tert null samples (Fig. 1A). The previously reported telomerase RNA template AtTER1 and the TERT-associated RNA AtTER2 were not found among these AtTERT-associated RNAs. To address the possibility that AtTER1 was masked by other more abundant RNAs, we used more stringent conditions to purify active telomerase by size exclusion chromatography before RIP (Fig. 1B). Telomerase activity was detected by quantitative telomere repeat amplification protocol (qTRAP) with the peak activity in a fraction corresponding to an apparent molecular mass of ∼300 kDa (Fig. 1C). A scatterplot of RNAs purified and sequenced from fractions with peak telomerase activity revealed a single RNA that was enriched more than 100-fold above background (Fig. 1D). This is the same RNA independently reported by Fajkus et al. (34) and dubbed AtTR. Since AtTER1 overlaps with the 5′ region of RAD52 locus (36) (SI Appendix, Fig. S1A), we performed additional TERT RIP experiments to directly test whether RAD52 mRNA was present in the IP. While RAD52 mRNA could be amplified from the IP, an RNA corresponding to the previously described AtTER1 could not (SI Appendix, Fig. 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.

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Fig. 1.

A single RNA species is enriched in active telomerase complexes. (A) Scatterplot representing RNA targets enriched in a direct RIP-seq experiment. WT and tert null mutant samples are compared to identify potential AtTERT-associated RNAs, labeled as blue or red according to their relative enrichment in WT greater than 2-fold or 5-fold, respectively. (B) Schematic representation of the experimental design for identification of telomerase-associated RNAs. (C) Size exclusion chromatogram of A. thaliana protein lysate. The blue curve shows the elution profile, and the red bars show the relative telomerase activity from each fraction. (D) Scatterplot of RNAs copurified with the active A. thaliana telomerase complex. AtTR is the only RNA molecule significantly enriched in WT samples compared with tert mutants.

AtTR Is Required for Telomere Repeat Synthesis by A. thaliana Telomerase.

AtTR was originally described as a noncoding Pol III transcript involved in the stress response (37). AtTR bears a 9-nt sequence of 5′-CUAAACCCU-3′ complementary to the A. thaliana 7-nt telomeric DNA sequence (TTTAGGG)_n (38). Mapping of its 5′ and 3′ ends by rapid amplification of cDNA ends revealed that AtTR is 268 nt long (SI Appendix, Fig. S2A). The size of endogenous AtTR was verified by Northern blot analysis (SI Appendix, Fig. S2B). Using direct terminator exonuclease treatment in combination with pyrophosphohydrolase, we found that AtTR bears a 5′ triphosphate structure (SI Appendix, Fig. S2C). AtTR is widely expressed but is most abundant in actively dividing cell culture. Notably, AtTR is also abundant in mature leaves, where AtTERT is conspicuously absent and telomerase activity is negligible (SI Appendix, Fig. S2D).

We used 2 genetic approaches to determine whether AtTR is required for telomerase activity and telomere maintenance in vivo. First, we found that a homozygous T-DNA insertion allele of AtTR (Flag_410H04) completely abolished AtTR RNA production as well as telomerase activity detected by qTRAP, while plants bearing a heterozygous mutation had ∼50% of the WT level of AtTR and 50% of the WT telomerase qTRAP activity (Fig. 2A and SI Appendix, Fig. S3 A and B). Terminal restriction fragment (TRF) analyses showed progressive shortening of the telomere tract in homozygous Flag_410H04 mutants over 5 generations (Fig. 2B), reminiscent of tert null mutants (39). Second, 2 independent CRISPR-mediated deletions that remove either a 49-nt sequence including the template or a 14 nt sequence downstream of the template disrupted telomere maintenance (SI Appendix, Fig. S4). We performed genetic complementation experiments on Flag_410H04 AtTR null mutants using an AtTR construct driven by the U6 promoter (U6::AtTR). Transformants with U6::AtTR expression exhibited restored telomerase activity and increased telomere length (Fig. 2C and SI Appendix, Fig. S3 C and D). These findings confirm that AtTR is necessary for both telomerase enzyme activity and telomere maintenance in A. thaliana.

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Fig. 2.

AtTR is the RNA template for Arabidopsis telomerase. (A, Top) Schematic representation of the AtTR gene showing the template domain (black box) and the location of a T-DNA insertion. (A, Bottom) Relative telomerase activity of WT, heterozygous, and homozygous AtTR mutants determined by quantitative TRAP assay. (B) TRF analysis of telomere length in AtTR mutants across multiple generations. (C) TRF results for genetic complementation with AtTR driven by the U6 promoter. Third-generation AtTR^−/− mutants were untransformed or transformed with U6::AtTR. (D) In vitro reconstitution of A. thaliana telomerase activity. Sequences of the putative template with the annealing position of 7 circular permuted telomeric DNA primers are shown on the right. The predicted primer-extended products are shown in red. A. thaliana telomerase is reconstituted in vitro from synthesized FLAGx3-AtTERT and 1.5 µM of T7 transcribed full-length AtTR (268 nt). The affinity-purified telomerase was assayed for activity in the presence of ³²P-dGTP, dTTP, dATP, and 7 plant telomeric DNA primers with permuted sequences. A radiolabeled 18-mer recovery control (r.c.) was added before product purification and precipitation. Numbers to the right of the gel denote the number of nucleotides added to the primer. (E) Template-directed nucleotide addition by A. thaliana telomerase. Telomerase was reconstituted in vitro with AtTERT and either AtTR_WT or AtTR_Hum. The reconstituted telomerase was assayed for activity in the presence of ³²P-dGTP and different combinations of dTTP, dATP, ddTTP, or ddATP. A 21-nt plant telomeric DNA primer (GTTTAGG)₃ was used for AtTR, and an 18-nt human telomeric DNA primer (GTTAGG)₃ was used for the AtTR_hum. A radiolabeled 18-mer recovery control was added before product purification and precipitation. Numbers and sequences of nucleotides added to the primers are indicated.

AtTR and AtTERT Reconstitute Active Telomerase In Vitro.

We next asked whether AtTR can assemble with AtTERT in vitro to reconstitute active telomerase. As shown in Fig. 2D, recombinant FLAGx3-AtTERT protein synthesized in rabbit reticulocyte lysate was assembled with T7 RNA polymerase transcribed AtTR in vitro and the reconstituted telomerase was immunopurified, followed by a direct primer extension assay (Fig. 2D). Seven A. thaliana telomeric DNA primers with permuted sequences of TTTAGGG bearing different 3′ terminal sequences were examined using in vitro reconstituted telomerase enzyme. The reaction with (GTTTAGG)₃ generated a 7-nt ladder pattern of products with major bands at positions +6, +13, and +20 (Fig. 2D, lane 8), consistent with the 7-nt telomeric DNA repeats synthesized by A. thaliana telomerase. A. thaliana telomerase exhibited similar levels of activity with the different permuted telomeric DNA primers and generated the expected offset banding patterns (Fig. 2D, lanes 2 to 7), indicating correct primer-template alignment and specific use of the template. Importantly, the primer extension activity is AtTR-dependent, as no activity was detected in the absence of AtTR (Fig. 2D, lane 1).

To further examine the templating function of AtTR, we generated an AtTR template mutant (AtTR_hum) with a template sequence similar to the human TR (hTR) template that allows the synthesis of 6-nt TTAGGG repeats. The telomeric TTAGGG repeats are ubiquitously conserved in most lineages of eukaryotes (3). The 9-nt AtTR template sequence 5′-CUAAACCCUGAACC-3′ for the synthesis of 7-nt repeats (TTTAGGG)n is flanked by a G residue at it 3′ boundary and potentially could be expanded to a longer 14-nt template by mutating the G residue to A. To convert the native A. thaliana template sequence to a human-like template, we simply deleted 1 A residue in the polymerization template sequence and the nonconserved G residue in the alignment sequence, which resulted in a 12-nt 5′-CUAACCCUAACC-3′ template for synthesizing TTAGGG repeats (Fig. 2E). As expected, the telomerase reconstituted from the AtTR_hum template mutant generated the first major band at position +5(+gttag) and the second major band at +11, indicating the addition of a 6-nt DNA repeat using the human-like template (Fig. 2E, lane 8). Moreover, the inclusion of dideoxy-ribonucleotides, either ddTTP or ddATP, terminated the primer extension reaction at the expected positions on the template of the AtTR_WT and AtTR_hum (Fig. 2E, lanes 2–3 and 6–7). In addition, under processive conditions with all 3 nucleotides, the AtTR_hum template with a long 6-nt alignment region led to a significantly higher processivity based on the ratio of +11/+5 products (Fig. 2E, lanes 4 and 8), consistent with a previous finding that longer templates correlate with higher repeat addition processivity (40). Taken together, these data demonstrate that the template sequence 43-CUAAACCCU-51 within AtTR is a bona fide template for telomeric DNA repeat synthesis by A. thaliana TERT.

Plant TRs Share a Conserved Secondary Structure.

To discern the structure of AtTR, we used phylogenetic comparative analysis to infer a secondary structure model from the sequence alignment of plant TR homologs identified from 3 major clades of land plant species: angiosperms, gymnosperms, and lycophytes (Fig. 3A). Orthologs of AtTR were identified by searching genomic sequence data predominantly from the National Center for Biotechnology using sequence homology search tools including BLAST, Fragrep2 (41), and Infernal (42). While the BLAST search was able to find TR homologs from closely related species, Fragrep2 allowed identification of TR homologs from more distantly related species by using position-specific weight matrix (PWM)-based searches with PWMs derived from multiple sequence alignments, as opposed to using the primary sequence as the search query. Collectively, we identified 85 AtTR orthologs, including 70 from angiosperms, 11 from gymnosperms, and 4 from lycophytes (SI Appendix, Table S1). To infer secondary structure, multiple sequence alignment analysis was performed with 16 representative TR sequences (12 angiosperms, 3 gymnosperms, and 1 lycophytes) selected from the 85 sequences to allow at least 1 representative from each individual order spanning 3 distinct clades (SI Appendix, Fig. S5). All TR sequences, including those from the basal groups, gymnosperms, and lycophytes, can be reliably aligned with the TR sequence from angiosperms, revealing universally conserved structural elements of plant TRs. From the alignment of 16 divergent plant TR sequences, universal or group-specific nucleotide covariations were identified to infer base-paired structural elements (Fig. 3 B–D and SI Appendix, Fig. S6). Comparison of TR secondary structures from 3 representative species—A. thaliana from angiosperms, Picea glauca (spruce) from gymnosperms and Selaginella kraussiana (spikemoss) from lycophytes—revealed 3 common structural features: a conserved T-PK core domain enclosed by stem P1c; a long stem comprising consecutive short base-paired regions termed P4, P5, and P6; and a long-range base-paired stem P1a formed between the extreme distal 5′ and 3′ sequences (Fig. 3 B–D).

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Fig. 3.

Plant TRs share a conserved secondary structure. (A) Evolutionary relationship between major land plant clades. A single representative species of each order is included. An asterisk denotes the species with the secondary structure models shown in B, C, and D. Representative TR secondary structures determined by phylogenetic sequence analysis are shown for A. thaliana from angiosperms (B), P. glauca (spruce) from gymnosperms (C), and S. kraussiana (spike moss) from lycophytes (D). The characteristic TR PK is shaded in yellow. Universal covariations (green line), group-specific covariations (blue line), and plant invariant residues (red) are indicated and based on sequence alignment of 16 divergent plant species spanning 8 eudicots, 2 monocots, 2 early-branching angiosperms, 3 gymnosperms, and 1 lycophyte. The aligned sequences are shown in SI Appendix, Fig. S5. (E) In vitro chemical probing of AtTR secondary structure by SHAPE. Chemical reactivities per nucleotide are plotted on the AtTR secondary structure. (F) In vivo chemical probing of AtTR structure by DMS-MaPseq. Average mutation frequencies per nucleotide are plotted on the AtTR secondary structure.

The plant T-PK core domain resembles those from ciliate, fungal, and vertebrate TRs consisting of a template, a universal PK structure formed by stems P2 and P3, and a core-enclosing stem P1c (Fig. 3 B–D). However, the plant T-PK core domain contains additional plant-specific stems, namely P1.1 (in P. glauca and S. kraussiana), P2.1 (in A. thaliana and P. glauca), and P2.2 (in P. glauca and S. kraussiana) (Fig. 3 B–D). The P1.1 stem can be found in the invertebrate echinoderm and fungal TRs, and could potentially function as a TBE (17, 43). The P2.1 and P2.2 stems are not present in all plant TRs, suggesting that they are more adaptable and may be important for a function specific to some plant groups. One possible role for the variable P2.1 and P2.2 stems is to maintain the length of the linker between the template and the PK structure within the T-PK core domain.

In addition to the T-PK core domain, the plant TR contains a long helical structure with 3 consecutive short stems—P4, P5, and P6—located near the 3′ end between P1a and P1b (Fig. 3 B–D). The location and structure of the plant P4/P5/P6 stem resembles the vertebrate CR4/5 domain, echinoderm eCR4/5 domain, or ciliate helix IV, all of which are essential for telomerase activity (28, 29, 43). The 3-way junction formed among P1a, P1b, and P4/5/6 appears to be a conserved feature of plant TR (Fig. 3 B–D). This P1a-mediated 3-way junction is unique to plant TR and is not found in other known TRs.

This conserved secondary structure model of AtTR is supported by chemical modification probing analysis. Selective 2’-hydroxyl acylation analysis by primer extension (SHAPE) was used to examine the accessibility of each nucleotide in the in vitro folded RNA (44). N-methylisatoic anhydride modification of individual nucleotides was monitored, and SHAPE activity plotted on the structural model to identify unpaired residues (Fig. 3E and SI Appendix, Fig. S7). Consistent with our AtTR structural model, most unpaired nucleotides showed significant SHAPE activity.

We also probed the AtTR structure in vivo by dimethyl sulfide (DMS) footprinting and mutational profiling (DMS-MaPseq). DMS methylates the base-pairing faces of single-stranded, unprotected adenosines and cytidines. Such modifications cause the stalling of conventional reverse transcriptases during cDNA synthesis, allowing for footprinting studies. These modifications can also result in mismatches in cDNA when TGIRT reverse transcriptase is used (45). DMS modifications were analyzed by primer extension (DMS footprinting), while DMS-induced mutational rates per position were calculated by coupling TGITR cDNA synthesis with high throughput sequencing. DMS footprinting identified 38 accessible nucleotides that mapped to predicted single-stranded residues (SI Appendix, Fig. S8). Results of DMS MaPseq extended these findings and revealed a detailed map of nucleotide accessibility (Fig. 3F and SI Appendix, Fig. S9). Accessible nucleotides were concentrated in the predicted single-stranded regions within the T-PK and P1b-P1c linker. Taken together, these in vitro and in vivo structural probing results provide strong support for our AtTR secondary structure model.

In addition to inferring the conserved secondary structure, the multiple sequence alignment of the 16 representative plant TRs spanning land plant evolution revealed 5 highly conserved regions (CRs), CR1 to CR5, containing nucleotides that are invariant among these 16 distantly related species (SI Appendix, Fig. S5). Such remarkable conservation of nucleotide identity usually predicts essential functions of these regions, as evident in vertebrate TRs (12). CR1 corresponds to the template of AtTR. CR2 and CR3 form the universal P2 and P3 stems of the PK, while CR4 and CR5 form a P5 structural element that includes the short 3-bp P5 stem, an asymmetric internal loop and the upper part of stem P4 (Fig. 3 B–D). While lacking the P6.1 stem-loop, the universal P5 structural element of the plant TR resembles the CR4/5 domain conserved in vertebrate, fission yeast, and filamentous fungal TRs (17, 28). This highly conserved P5 stem may serve as a protein-binding site or play a crucial role in telomerase function.

The AtTR PK Domain Is Essential for Telomerase Function and Homologous to Human TR.

With a robust secondary structure model for AtTR, we sought to map the structural elements essential for telomerase activity. Full-length or truncated AtTR constructs were assembled with recombinant FLAGx3-AtTERT in vitro, and the immunopurified enzymes were analyzed for telomerase activity by direct primer extension. Analysis of 3 truncated AtTR fragments—11 to 179, 25 to 153, and 42 to 136 (Fig. 4A)—showed that AtTR-25 to 153 is the minimal PK fragment sufficient to reconstitute ∼40% of WT activity without the P4/5/6 domain (Fig. 4B, lanes 2 and 3). The core-enclosing P1c stem appeared to be important for telomerase function, as the AtTR-42–136 fragment with P1c removed was unable to reconstitute any significant activity (Fig. 4B, lane 4). Equivalent to the CR4/5 domain of human TR, the 3′ P1a/4/5/6 domain of AtTR can also function in trans as a separate RNA molecule to stimulate the reconstituted activity from the basal 40% to 66% of the WT level (Fig. 4C). A basal activity of telomerase reconstituted from the T-PK domain alone was previously reported with Trypanosome and Echinoderm TRs (31, 43), indicating an evolutionary transition of functional dependence for the 2 conserved TR domains.

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Fig. 4.

Functional characterization of critical structural elements in AtTR. (A) Schematic of the AtTR secondary structure. The 5′ and 3′ residues of truncated AtTR fragments are denoted on the AtTR structure. The positions and identities of specific point mutations introduced are indicated. (B) Identification of a minimal PK fragment and (C) functional analysis of stem P1a/4/5/6. Full-length AtTR (AtTR-FL) and various AtTR truncated fragments were assembled with AtTERT in vitro and analyzed for activity by a primer extension assay. The number of nucleotides (+6, +13 or +20) added in each major band of product are indicated. The P1a/4/5/6 fragment was generated by deleting residues 25 to 153 from the AtTR-FL and replacing them with a GAAA tetraloop. The relative activities of the reactions are indicated under the gel. A recovery control (r.c.) is shown. (D) The functional requirement of invariant U residues in PK domain. (E) The effect of P1c linker length on template boundary definition. (F) Compensatory mutagenesis analysis of stem P5. (G) Compensatory mutagenesis analysis of stem P2.1. AtTR-FL constructs bearing specific point mutations are assembled with AtTERT in vitro and analyzed for telomerase activity. For analyzing template boundary definition with AtTR-38UU, the reconstituted enzyme was analyzed in the absence (−) or presence (+) of dCTP in addition to dGTP, dATP, and dTTP.

The PK structure of plant TRs highly resembles the PK structures in ciliate and vertebrate TRs with differences in size and complexity. In the human TR PK structure, the invariant U residues in the J2/3 upstream region (J2/3u) are essential for telomerase activity (46). To determine whether the invariant U residues in plant TR PK are functionally homologous to the human TR, we reconstituted telomerases with two AtTR mutants, U92C and UU94/95CC. The activity assays of the mutant enzyme showed no activity (Fig. 4D, lanes 2 and 3), indicating that these U residues in the AtTR PK domain are absolutely required for telomerase activity. Therefore, the T-PK domains of AtTR and hTR are both structurally and functionally homologous.

Another critical function provided by the T-PK domain is defining the functional template boundary through specific structural elements, that is, the P1 stem in vertebrate TR (20). The P1c stem in the T-PK domain of AtTR resembles the P1 stem in human TR and presumably functions as the template boundary element. To test this idea, we generated an AtTR mutant 38UU with 2 U residues inserted between the P1c stem and the template to increase the linker length, a critical determinant of the template boundary. In the WT AtTR template, a G residue immediately flanks the 5′ boundary and does not serve as a template even in the presence of dCTP substrate (Fig. 4E, lanes 1 and 2). However, in the presence of dCTP, the telomerase enzyme reconstituted with the AtTR mutant 38UU uses the G residue as a template beyond the template boundary (Fig. 4E, lanes 3 and 4). Thus, A. thaliana and human telomerases share a homologous mechanism for template boundary definition.

While the overall secondary structure of AtTR is well supported by covariation evidence and chemical probing data, we performed mutagenesis analysis to provide additional support for the highly conserved P5 stem and the plant-specific P2.1 stem (Fig. 4A). The 3-bp P5 stem is formed by 2 highly conserved regions, CR4 and CR5, with only limited covariation support for 1 of the 3 base pairs. We thus generated AtTR full-length constructs, P5-m1 and -m2, with 2 single point mutations, G194C and C239G, introduced to disrupt the invariant G:C base-pairing in the P5 stem, or a compensatory mutant P5-m3 with both point mutations to restore the base-pairing (Fig. 4A). The activity assay showed that P5-m1 and -m2 single point mutations abolished telomerase activity (Fig. 4F, lanes 2 and 3), while the compensatory mutation P5-m3 restored activity (Fig. 4F, lane 4), consistent with the essential base-paired structure of stem P5. A similar mutagenesis approach was used to confirm the base-paired structure and the functional importance of stem P2.1 (Fig. 4G). Taken together, these in vitro studies strongly support the robustness of the phylogenetic comparative analysis for inferring RNA secondary structure in plant TR.