A flexible template boundary element in the RNA subunit of fission yeast telomerase

Abstract

Telomerase adds telomeric repeat sequences to chromosome ends using a short region of its RNA subunit as a template. Telomerase RNA subunits are phylogenetically highly divergent, and different strategies have evolved to demarcate the boundary of the template region. The recent identification of the gene encoding telomerase RNA in the fission yeast Schizosaccharomyces pombe (ter1+) has opened the door for structure-function analyses in a model that shares many features with the telomere maintenance machinery of higher eukaryotes. Here we describe a structural element in TER1 that defines the 5' boundary of the template. Disruption of a predicted long range base pairing interaction between template-adjacent nucleotides and a sequence further upstream resulted in reverse transcription beyond the template region and caused telomere shortening. Normal telomere length was restored by combining complementary nucleotide substitutions in both elements, showing that base pairing, not a specific sequence, limits reverse transcription beyond the template. The template boundary described here resembles that of budding yeasts and some mammalian telomerases. However, unlike any previously characterized boundary element, part of the paired region overlaps with the template itself, thus necessitating disruption of these interactions during most reverse transcription cycles. We show that changes in the paired region directly affect the length of individual telomeric repeat units. Our data further illustrate that marginal alignment of the telomeric 3' end with RNA sequences downstream of the template is responsible for primer slippage, causing incorporation of strings of guanosines at the start of a subset of repeats.

FIGURE 1.

Determination of the 5′ end of TER1 by primer extension. Oligonucleotide BLoli1116 (complementary to nucleotides 98-118) was radiolabeled and used for primer extension with total RNA, poly(A) enriched fraction, and RNA isolated from Trt1-Myc immunopurifications. No signal was observed with total RNA from a ter1Δ strain (lane 7). The three major 5′ ends are marked with arrows. The percentages next to the sequence indicate relative amount of RNA isolated from the Trt1-Myc sample starting at this position. Dideoxynucleotide sequencing (lanes 1-4) was performed with BLoli1116 and a cloned fragment of the ter1⁺ gene. Sequencing lanes are labeled for the strand representing the TER1 RNA.

FIGURE 2.

Disruption of the template boundary element causes telomere shortening. A, schematic of template (green) and boundary element. Structure predictions on nucleotides 1-1213 were performed with Mfold. Two alternative local structures are shown. B, schematic of mutations designed to test the predicted helix adjacent to the template. Mutated nucleotides are in red in DMA, DMB, and the CM. Telomeric DNA sequence complementary to the template is shown in blue, and two nucleotides of read-through product are underlined. C, telomeric Southern blot for ter1^- cells harboring vector only (lane 1) or plasmids with wild type ter1⁺ (lane 2) or mutants as indicated (lanes 3-8). Genomic DNA was digested with EcoRI, separated on a 1% agarose gel, transferred to a nylon membrane, and probed with the nick translation products from a 300-bp telomeric DNA fragment. A probe against the rad16⁺ gene was used as loading control (LC). D, TER1 levels from wild type (lane 1), DMA (lane 2), DMB (lane 3), and CM (lane 4) were analyzed by Northern blotting of total RNA samples (15 μg/lane). The small nucleolar RNA snR101 was used as loading control (LC).

FIGURE 3.

Boundary element disruption mutants result in extended reverse transcription products in vitro and in vivo. A, in vitro activity assay for wild type (lane 1), DMA (lane 2), DMB (lane 3), and CM (lane 4). Telomerase assays were carried out as described in Ref. . A 100-mer oligonucleotide was used as loading control (LC). A schematic for the alignment of the telomeric primer (blue) upstream of the template (green) is shown above the gel. Nucleotides added by telomerase are shown to the left of the gel. B, analysis of cloned telomere sequences from wild type (wt, n = 141), DMA (n = 83), and DMB (n = 79). Telomeres were isolated after 80 generations in the presence of the ter1 mutant, cloned by G overhang capture assay and sequenced. After trimming of the invariant proximal part of each telomere, the relative abundance of the three most common repeats was determined. C, sequences for the distal part of four telomeres isolated from DMA mutant cells. Read-through products are highlighted in bold, capital letters. Aberrant sequences found in only one telomere are underlined.

FIGURE 4.

An extended boundary helix causes shortening of telomeric repeat units. A, schematic of the template and boundary element region in the ter1-17 mutant. The template is in green, and mutated nucleotides are shown in red. An arrow underneath the unpaired core template nucleotides denotes the direction of reverse transcription. The energetically favored structure of this region was determined by Mfold. B, Southern blot of EcoRI-digested genomic DNA from wild type (wt) and ter1-17 mutant cells probed with a telomeric fragment. A probe against the rad16⁺ gene was used as loading control (LC). C, a graphical representation of the distribution of different telomeric repeats in wild type (n = 141) and ter1-17 (n = 72) cells 80 generations after the introduction of mutant telomerase.

FIGURE 5.

Longer repeat units in the presence of a destabilized boundary element. A, schematic of template and boundary element of TER1 with boundary element mutation C232A (ter1-3). The mutated nucleotide is shown in red. Structures were determined by Mfold, and two alternative folds are shown. B, graphical representation of the relative abundance of telomeric repeat units within the variable part of telomeres after 80 generations. The number of telomeres (n) in each data set was 141 (wild type), 72 (ter1-3), 77 (ter1-36), 72 (ter1-31), and 71 (ter1-34). C, template and boundary element structures for U231C (ter1-36). D, schematics of local structures for ter1-31 and ter1-34. E, telomerase activity assay for wild type and ter1 mutants. For lane 2, telomerase was incubated with RNase A (5 ng/μl) for 10 min prior to the addition of primer and nucleotides. The +1 position is indicated on the right, and nucleotides added by telomerase are on the left of the gel. wt, wild type.

FIGURE 6.

Effect of replacing the template-proximal A-U base pair with G-C. A, schematic of ter1-18 mutation. B, graph depicting the relative abundance of different repeat units. GGTTACCG and GGTTACCCG repeats are not present in wild type telomeres. As the part of ter1-18 telomeres included in the analysis contained wild type GGTTACA and mutant GGTTACC repeats, the sum of both was plotted. GGTTACCC and GGTTACAC sequences were treated in the same manner. wt, wild type.

FIGURE 7.

G stuttering in relation to the previous repeat sequence. A, schematic of alignment for telomeric DNA ending in AC (left) or ACA (right). The template is shown in green, and the newly added telomeric sequence is in red. B, telomere data sets for wild type and mutants were processed with TweenMotif to reveal the relative abundance of two to nine guanosines following each of the five repeats shown below. The total number of repeats analyzed in each category is shown above the columns.