The transition from circular to linear chromosomes in eukaryotes introduced the “end-replication problem” which is the inherent inability of cellular DNA polymerases to completely replicate linear chromosomal ends. Over evolutionary time, eukaryotes evolved “caps” at their chromosomal ends which are DNA protein complexes known as telomeres. Although telomeric DNA does suffer from the incomplete end-replication, the telomerase ribonucleoprotein enzyme was evolved as the dominant and winning solution to this problem in eukaryotes. The protein component of telomerase known as Telomerase reverse transcriptase (TERT), is well conserved across broad eukaryotic groups. In contrast, the RNA component of telomerase, telomerase RNA (TR) is extremely divergent in terms of sequence and length. This presents insurmountable challenges in the identification of novel TR molecules, especially from more distant and previously unexplored eukaryotic groups. Although animal TRs have been identified and studied in detail, the early evolution and origins of animal telomerases remain a mystery. Thus, it is crucial to study telomerases from the earliest ancestors of animals. The Choanoflagellates are a group of free-living unicellular eukaryotes that are deemed to be the closes living relatives of animals. The choanoflagellate M. brevicollis (Mbr) is a model eukaryote used to study the origins of multicellularity. Thus, we determined to purify M. brevicollis telomerase to isolate, sequence and identify the co-purifying TR. Towards achieving this ultimate goal, this study focuses on partially purifying M. brevicollis telomerase via polyethylene glycol (PEG) precipitation. As the first step, reliable and reproducible culture conditions for M. brevicollis were established. Following this, larger scale cell cultures were grown and used for PEG precipitation. Final concentrations of 5%, 10%, and 20% PEG were used. PEG precipitates were resuspended in buffer and quantitated using Bradford assay. PEG precipitated macromolecular complexes were subject to Western blot using custom generated anti-MbrTERT antibodies which revealed a clear band proximal in size to the 75 kDa marker consistent with the 87 kDa putative MbrTERT. This study serves as a launchpad for the identification of MbrTR towards delineating the early evolution of telomerase in animals.

Telomerase is a ribonucleoprotein (RNP) enzyme that requires an integral telomerase RNA (TR) subunit, in addition to the catalytic telomerase reverse transcriptase (TERT), for enzymatic function. The secondary structures of TRs from the three major groups of species, ciliates, fungi, and vertebrates, have been studied extensively and demonstrate dramatic diversity. Herein, we report the first comprehensive secondary structure of TR from echinoderms—marine invertebrates closely related to vertebrates—determined by phylogenetic comparative analysis of 16 TR sequences from three separate echinoderm classes. Similar to vertebrate TR, echinoderm TR contains the highly conserved template/pseudoknot and H/ACA domains. However, echinoderm TR lacks the ancestral CR4/5 structural domain found throughout vertebrate and fungal TRs. Instead, echinoderm TR contains a distinct simple helical region, termed eCR4/5, that is functionally equivalent to the CR4/5 domain. The urchin and brittle star eCR4/5 domains bind specifically to their respective TERT proteins and stimulate telomerase activity. Distinct from vertebrate telomerase, the echinoderm TR template/pseudoknot domain with the TERT protein is sufficient to reconstitute significant telomerase activity. This gain-of-function of the echinoderm template/pseudoknot domain for conferring telomerase activity presumably facilitated the rapid structural evolution of the eCR4/5 domain throughout the echinoderm lineage. Additionally, echinoderm TR utilizes the template-adjacent P1.1 helix as a physical template boundary element to prevent nontelomeric DNA synthesis, a mechanism used by ciliate and fungal TRs. Thus, the chimeric and eccentric structural features of echinoderm TR provide unparalleled insights into the rapid evolution of telomerase RNP structure and function.

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.