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.

Telomeres are repetitive DNA sequences at linear chromosome termini, protecting chromosomes against end-to-end fusion and damage, providing chromosomal stability. Telomeres shorten with mitotic cellular division, but are maintained in cells with high proliferative capacity by telomerase. Loss-of-function mutations in telomere-maintenance genes are genetic risk factors for cirrhosis development in humans and murine models. Telomerase deficiency provokes accelerated telomere shortening and dysfunction, facilitating genomic instability and oncogenesis. Here we examined whether telomerase mutations and telomere shortening were associated with hepatocellular carcinoma (HCC) secondary to cirrhosis. Telomere length of peripheral blood leukocytes was measured by Southern blot and qPCR in 120 patients with HCC associated with cirrhosis and 261 healthy subjects. HCC patients were screened for telomerase gene variants (in TERT and TERC) by Sanger sequencing. Age-adjusted telomere length was comparable between HCC patients and healthy subjects by both Southern blot and qPCR. Four non-synonymous TERT heterozygous variants were identified in four unrelated patients, resulting in a significantly higher mutation carrier frequency (3.3%) in patients as compared to controls (p = 0.02). Three of the four variants (T726M, A1062T, and V1090M) were previously observed in patients with other telomere diseases (severe aplastic anemia, acute myeloid leukemia, and cirrhosis). A novel TERT variant, A243V, was identified in a 65-year-old male with advanced HCC and cirrhosis secondary to chronic hepatitis C virus (HCV) and alcohol ingestion, but direct assay measurements in vitro did not detect modulation of telomerase enzymatic activity or processivity. In summary, constitutional variants resulting in amino acid changes in the telomerase reverse transcriptase were found in a small proportion of patients with cirrhosis-associated HCC.

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.