Telomere les(i/s)ons from a telomerase RNA mutant

The “end replication problem,” the inability of conventional DNA polymerases to duplicate the very ends of linear DNA molecules, is usually solved by telomerase, a specialized reverse transcriptase. The telomerase holoenzyme consists of the catalytic subunit (TERT), a telomerase RNA (TER) and species-specific accessory factors. Although telomerase RNAs vary enormously in size and nucleotide sequence, 4 structures are highly conserved: (1) a telomere repeat templating sequence, which is single-stranded in the folded RNA; (2) a template boundary element (TBE) helix that is immediately adjacent to the template and prevents synthesis past the template; (3) a pseudoknot that affects template usage, and (4) the stem terminus element (STE), which was discovered in mammals in 2000 where it is essential for telomerase activity in vitro and in vivo yet its mode of action was previously unknown. Except for the STE, the 3 other elements are in the catalytic core region of the RNA. This review summarizes a recent paper that takes advantage of the conservation of STE sequence and structure in the fission yeast Schizosaccharomyces pombe (Fig. 1) to reveal the function of the STE. To understand how the STE works, we generated a panel of fission yeast STE mutants and determined their effects on telomere length. This characterization showed that, as in mammals, the fission yeast STE is essential for telomerase activity and telomere maintenance and identified a partial-loss-of-function allele, ter1-STEloop. In this mutant, the 4 tandem uridines in the STE tetraloop are changed to adenines (Fig. 1). Because telomeres are short but stable in ter1-STEloop cells, the mutant was ideal for analyzing STE function in vivo. Although non-canonical telomere repeats are seen in most organisms, fission yeast telomeric DNA is particularly heterogeneous, as reflected in its consensus sequence, 50-(G)0–6GGTTACAC-30 (the rare cytosine, discussed below, is underlined). Our ability to isolate the ter1-STEloop allele was probably facilitated by this heterogeneity, which arises from the inefficiency of the fission yeast TBE, which is less capable than TBEs in other organisms at preventing run-on reverse transcription. In wild type fission yeast, occasional TBE failure results in reverse transcription into the TBE, which introduces a rare cytosine at the end of the core telomere sequence in about 12% of repeats. The variable number of guanine residues is generated by “stuttering” that arises from the absence of base-pairing between the RNA template and the telomeric DNA end. Because incorporation of the rare cytosine changes the template RNA-telomeric DNA register, its addition results in reduced stuttering and hence fewer guanine-tracts (Reviewed in). Although we considered that the STE promotes holoenzyme formation, TERT association with TER1 was unaffected in ter1-STEloop cells. Likewise, the physical interaction between telomerase and the telomere was normal. However, as in humans, telomerase catalysis is compromised as extracts from ter1-STEloop cells had very low activity. We isolated and cloned telomeric DNA from mutant and wild type cells and determined their sequence. For cloning, we developed a novel method that exploited the rapid but reversible telomere shortening that occurs by growing fission yeast cells at higher temperatures. This approach ensured that we analyzed only the sequence templated by TER1-STEloop. Surprisingly, ter1-STEloop telomeric DNA does not have wild type sequence. Rather, the mutant telomeres have a much higher frequency of rare cytosines and reduced frequency of guanine tracts, 2 phenotypes first seen in cells with mutant TBEs (Fig. 1). The unusual sequence of ter1-STEloop telomeric DNA was recombination independent but telomerase dependent. Thus, the STE affects how the telomerase catalytic core templates nucleotide addition. Because the ter1-STEloop mutation affects the sequence of telomeric DNA in the same way as mutations that disrupt TBE function, we conclude that the STE enforces TBE activity. The TER1-STEloop templated change in telomeric sequence also affected telomeric chromatin. In fission yeast and mammals, telomeres are bound by the multi-subunit shelterin complex, which together with the telomeric DNA, protects telomeres and mediates all telomere functions. Using chromatin immunoprecipitaton, we analyzed telomere binding of