Fig 1.
Analysis of TER1 and TER2 loci across A. thaliana accessions.
(A) Schematic diagram of TER1, TER2, and TER2S. TER1 and TER2 share a core region of ~219 nt comprised of conserved regions 1 and 2 (CR1 and CR2). The telomere template is denoted by a vertical black bar in CR1. TER2S is formed by splicing to remove the DSB responsive element (DRE) and elimination of the 3’ terminus (3’ R). (B) Analysis of TER1 among 853 A. thaliana accessions. Identity shown in green denotes regions 100% nucleotide similarity whereas mustard yellow indicates variation. There is one colored line for each nucleotide. The height of the bar indicates the degree of variation. Percent identity for each region is denoted in % above each RNA region or for the entire RNA to the right. The telomere template region is indicated by the horizontal black bar. (C) Analysis of TER2 in 853 accessions. Color scheme is the same as in (B). Asterisk indicates that for percent identity to be calculated in a given region, sequence data must be present in all accessions. Sequence was missing for DRE and CR2 for some accessions, and hence these regions are listed as having 0% ID. However, >60% of the accessions were 100% conserved in DRE, and 98% of accessions were 99% conserved in CR2. See S2 Fig for complete alignment of TER2 in 853 accessions.
Fig 2.
The TER2 intervening sequence has the properties of a Copia-like solo LTR.
(A) Schematic of the five chromosomes in A. thaliana Col-0 illustrating the locations of TER1, TER2 and DRE on the left arm of chromosome 3 (DRE3L) and the right arm of chromosome 3 (DRE3R) (schematic adapted from TAIR). (B) Phylogenetic tree of select Brassicaceae members (including the Brassicales member Carica papaya). The number of solo and full-length DREs identified by BLAST are shown to the right. Approximate time of divergence was adapted from [29]. Representative A. thaliana accessions re indicated by the triangle. (C) Sequences at the 5’ and 3’ boundary elements of DRE in TER2 (top), DRE3L (middle), and DRE3R (bottom) are shown. Nucleotides within the target site duplication are denoted by the green bar and tandem inverted repeats of DRE are represented by the brown bar.
Fig 3.
Expression of TER2Δ and association with TERT.
(A) RT-PCR results for TER2Δ in Ler-0 and TER2 in Col-0. Primer positions are indicated by arrows in panel B. (B) Schematic showing sequencing results for TER2 and TER2Δ PCR products from Col-0 and Ler-0 obtained from (A). The target site duplication is indicated by the green underlined nucleotides. Tandem inverted repeats are indicated by brown nucleotides. (C) qPCR results for TER1, TER2 and TER2Δ in Col-0 and Ler-0. For comparison, the Col-0 TER1 level was set to 1.
Fig 4.
DSB-mediated RNA induction and telomerase inhibition are associated with DRE.
(A) Table indicating the TER2 transcript status for four A. thaliana accessions. (B) and (C) show qPCR results for Col-0 and Ler-0 seedlings treated with zeocin for the time points indicated. Data for the BRCA1 control (B) and TER2 (Col-0) or TER2Δ (Ler-0) transcripts (C) are shown. (D) qPCR results for accessions with TER2 (Ws-2) and TER2Δ (Co-1) submitted to the zeocin regimen for 2 h. (E) qPCR results following TERT immunoprecipitation in Col-0 and Ler-0 seedlings treated with or without zeocin (time point). The TER2:TER1 ratio in Col-0 and the TER2Δ:TER1 ratio in Ler-0 are shown. Values were normalized to Col-0 TER2:TER1 ratio in the absence of zeocin (set to 1). (F) qTRAP results for Col-0 and Ler-0 seedlings with or without zeocin treatment. (G) qTRAP results for the samples in (D) and the 2 h time point from (C). Telomerase activity was normalized to the corresponding untreated controls and set to 1. Red dashed bar indicates no change between treated and untreated samples. The changes in telomerase activity in Col-0 and Ws-2 were statistically significant (p-value< 0.05). Significance was calculated relative to untreated samples using a Student’s t-test. For all experiments, n > 3.
Fig 5.
TER2 not TER2Δ represses telomerase activity.
(A) qPCR results are shown for transgenic seedlings expressing TER2 in Ler-0 or TER2Δ in Col-0. TER1 and TER2 levels were normalized to the values in wild type Col-0 (set to 1). TER2Δ was normalized to the value in wild type Ler-0 (set to 1). GAPDH served as a reference gene. (B) qTRAP results are shown for the seedlings analyzed in (A). Relative telomerase activity was normalized to wild type Col-0. The change in telomerase activity in Ler-0 transformants expressing TER2 relative to wild type Ler-0 is statistically significant (p-value<0.005). Significance was calculated relative to untreated samples using a Student’s t-test. For all experiments, n > 3.
Fig 6.
TER2 is a labile RNA transcript stabilized by DNA damage.
qPCR results are shown for TER1 and TER2/TER2Δ from Col-0 and Ler-0 in the presence of cordycepin. Col-0 and Ler-0 seedlings were treated with cordycepin (100μg/μl) for the times indicated followed by qPCR to monitor TER1 (A) and TER2/ TER2Δ (B). The values obtained for untreated RNA samples were set to 0 and the fold decrease is shown. eIF-4a was used as reference gene for normalization. (C-E) qPCR results from a time course experiment of Col-0 seedlings treated with cordycepin followed by zeocin. Seedlings were incubated with cordycepin for 1.5 h to shut down transcription, and zeocin was added (red arrows). The incubation continued for a total of 3.5 h. Results for BRCA1 (C), TER1 (D), TER2 (E) are shown.
Fig 7.
Model for exaptation of a TE into TER2 and the emergence of a telomerase regulatory lncRNA.
Duplication of the single copy ancestral TER gene was followed soon thereafter by exaptation of DRE into the A. thaliana TER2 locus. The majority of A. thaliana accessions retain DRE (e.g. Col-0), but a small subset lost it (e.g. Ler-0). TER2Δ is produced by accessions lacking DRE. DRE acts as a post-transcriptional sensor that modulates TER2 abundance in response to DNA damage. Under normal physiological conditions, TER2 is an unstable RNA. However, in the presence of DSBs, TER2 is rapidly induced. Whether this is due to direct RNA stabilization or inhibition of TER2 processing (to yield TER2s) is unknown. TER2 has a higher affinity for TERT than TER1 or TER2Δ and following induction by DNA damage accumulates in TERT containing complexes in vivo. TER2-mediated telomerase inhibition may reflect competitive inhibition of TER1 for TERT. The transient decrease in telomerase activity may promote DSB repair rather than de novo telomere formation, thereby stabilizing the genome.