Telomerase emerged during evolution as a prominent solution to the eukaryotic linear chromosome end-replication problem. Telomerase minimally comprises the catalytic telomerase reverse transcriptase (TERT) and telomerase RNA (TR) that provides the template for telomeric DNA synthesis. While the TERT protein is well-conserved across taxa, TR is highly divergent amongst distinct groups of species. Herein, we have identified the essential functional domains of TR from the basal eukaryotic species Trypanosoma brucei, revealing the ancestry of TR comprising two distinct structural core domains that can assemble in trans with TERT and reconstitute active telomerase enzyme in vitro. The upstream essential domain of T. brucei TR, termed the template core, constitutes three short helices in addition to the 11-nt template. Interestingly, the trypanosome template core domain lacks the ubiquitous pseudoknot found in all known TRs, suggesting later evolution of this critical structural element. The template-distal domain is a short stem-loop, termed equivalent CR4/5 (eCR4/5). While functionally similar to vertebrate and fungal CR4/5, trypanosome eCR4/5 is structurally distinctive, lacking the essential P6.1 stem-loop. Our functional study of trypanosome TR core domains suggests that the functional requirement of two discrete structural domains is a common feature of TRs and emerged early in telomerase evolution.

Telomerase RNA (TER) is an essential component of the telomerase ribonucleoprotein complex. The mechanism for TER 3′-end processing is highly divergent among different organisms. Here we report a unique spliceosome-mediated TER 3′-end cleavage mechanism in Neurospora crassa that is distinct from that found specifically in the fission yeast Schizosaccharomyces pombe. While the S. pombe TER intron contains the canonical 5′-splice site GUAUGU, the N. crassa TER intron contains a non-canonical 5′-splice site AUAAGU that alone prevents the second step of splicing and promotes spliceosomal cleavage. The unique N. crassa TER 5′-splice site sequence is evolutionarily conserved in TERs from Pezizomycotina and early branching Taphrinomycotina species. This suggests that the widespread and basal N. crassa-type spliceosomal cleavage mechanism is more ancestral than the S. pombe-type. The discovery of a prevalent, yet distinct, spliceosomal cleavage mechanism throughout diverse fungal clades furthers our understanding of TER evolution and non-coding RNA processing.

Telomerase is a specialized reverse transcriptase (RT) containing an intrinsic telomerase RNA (TR) component. It synthesizes telomeric DNA repeats, (GGTTAG)n in humans, by reiteratively copying a precisely defined, short template sequence from the integral TR. The specific mechanism of how the telomerase active site uses this short template region accurately and efficiently during processive DNA repeat synthesis has remained elusive. Here we report that the human TR template, in addition to specifying the DNA sequence, is embedded with a single-nucleotide signal to pause DNA synthesis. After the addition of a dT residue to the DNA primer, which is specified by the 49 rA residue in the template, telomerase extends the DNA primer with three additional nucleotides and then pauses DNA synthesis. This sequence-defined pause site coincides precisely with the helix paired region 1 (P1)-defined physical template boundary and precludes the incorporation of nontelomeric nucleotides from residues outside the template region. Furthermore, this sequence-defined pausing mechanism is a key determinant, in addition to the P1-defined template boundary, for generating the characteristic 6-nt ladder banding pattern of telomeric DNA products in vitro. In the absence of the pausing signal, telomerase stalls nucleotide addition at multiple sites along the template, generating DNA products with heterogeneous terminal repeat registers. Our findings demonstrate that this unique self-regulating mechanism of the human TR template is essential for high-fidelity synthesis of DNA repeats.