An intact putative mouse telomerase essential N-terminal domain is necessary for proper telomere maintenance.

BACKGROUND INFORMATION: Naturally occurring telomerase reverse transcriptase (TERT) isoforms may regulate telomerase activity, and possibly function independently of telomeres to modulate embryonic stem (ES) cell self-renewal and differentiation. RESULTS: We report the characterisation of two novel mouse TERT (mTERT) splice variants, Ins-i1[1-102] (Insi1 for short) and Del-e12[1-40] (Dele12 for short) that have not been previously described. Insi1 represents an in-frame insertion of nucleotides 1-102 from intron 1, encoding a 34 amino acid insertion at amino acid 73. Based on known functions of this region in human and Tetrahymena TERTs, the insertion interrupts the RNA interaction domain 1 implicated in low-affinity RNA binding and the telomerase essential N-terminal domain implicated in DNA substrate interactions. Dele12 contains a 40 nucleotide deletion of exon 12 which generates a premature stop codon, and possible protein lacking the C-terminus. We found Insi1 expressed in adult mouse brain and kidney and Dele12 expressed in adult mouse ovary. Dele12 was inactive in vitro and in mTERT(-/-) ES cells and Insi1 retained 26-48% of telomerase activity reconstituted by wild-type mTERT in vitro and in mTERT(-/-) ES cells. The Insi1 variant exhibited reduced DNA substrate binding in vitro and both variants exhibited a reduction in binding the telomerase RNA, mTR, when expressed in mTERT(-/-) ES cells. Stable expression of Dele12 in the mouse fibroblast CB17 cell line inhibited telomerase activity and slowed cell growth, suggesting a potential dominant-negative effect. Levels of signal-free ends, representing short telomeres, and end-to-end fusions were higher in mTERT(-/-) ES cells expressing mTERT-Insi1 and mTERT-Dele12, compared with levels observed in mTERT(-/-) ES cells expressing wild-type mTERT. In addition, in mTERT(-/-) cells expressing mTERT-Insi1, we observed chromosomes that were products of repeated breakage-bridge-fusion cycles and other telomere dysfunction-related aberrations. CONCLUSION AND SIGNIFICANCE: An intact mTERT N-terminus which contributes to mTR binding, DNA binding and telomerase activity is necessary for elongation of short telomeres and the maintenance of functional telomeres. It is reasonable to speculate that relative levels of mTERT-Insi1 may regulate telomere function in specific tissues.

Dyskeratosis congenita mutations in dyskerin SUMOylation consensus sites lead to impaired telomerase RNA accumulation and telomere defects.

Mutations in the dyskerin gene (DKC1) cause X-linked dyskeratosis congenita (DC), a rare and fatal premature aging syndrome characterized by defective telomere maintenance. Dyskerin is a highly conserved nucleolar protein, and a component of the human telomerase complex that is essential for human telomerase RNA (hTR) stability. However, its regulation remains poorly understood. Here, we report that dyskerin can be modified by small ubiquitin-like modifiers (SUMOs). We find that human DC-causing mutations in highly conserved dyskerin SUMOylation consensus sites lead to impaired hTR accumulation, telomerase activity and telomere maintenance. Finally, we show that modification of dyskerin by SUMOylation is required for its stability. Our findings provide the first evidence that dyskerin stability is regulated by SUMOylation and that mutations altering dyskerin SUMOylation can lead to defects in telomere maintenance that are characteristics of DC.

Regulation of telomere length and homeostasis by telomerase enzyme processivity.

Telomerase is a ribonucleoprotein consisting of a catalytic subunit, the telomerase reverse transcriptase (TERT), and an integrally associated RNA that contains a template for the synthesis of short repetitive G-rich DNA sequences at the ends of telomeres. Telomerase can repetitively reverse transcribe its short RNA template, acting processively to add multiple telomeric repeats onto the same DNA substrate. The contribution of enzyme processivity to telomere length regulation in human cells is not well characterized. In cancer cells, under homeostatic telomere length-maintenance conditions, telomerase acts processively, whereas under nonequilibrium conditions, telomerase acts distributively on the shortest telomeres. To investigate the role of increased telomerase processivity on telomere length regulation in human cells with limited lifespan that are dependent on human TERT for lifespan extension and immortalization, we mutated the leucine at position 866 in the reverse transcriptase C motif of human TERT to a tyrosine (L866Y), which is the amino acid found at the equivalent position in HIV-1 reverse transcriptase. We report that, similar to the previously reported gain-of-function Tetrahymena telomerase mutant (L813Y), the human telomerase variant displays increased processivity. Human TERT-L866Y, like wild-type human TERT, can immortalize and extend the lifespan of limited-lifespan cells. Moreover, cells expressing human TERT-L866Y display heterogenous telomere lengths, telomere elongation, multiple telomeric signals indicative of fragile sites and replicative stress, and an increase in short telomeres, which is accompanied by telomere trimming events. Our results suggest that telomere length and homeostasis in human cells may be regulated by telomerase enzyme processivity.

Quantifying candidate volume for endovascular therapy for acute ischemic stroke: a retrospective chart review.

BACKGROUND: Endovascular therapy is the new standard of care for certain patients with acute ischemic stroke. We aimed to determine whether procedural volumes at an academic health sciences centre in northeastern Ontario exceeded the minimum of 20 procedures annually to support establishment of an endovascular therapy centre and thus improve regional access to this type of care. METHODS: We conducted a retrospective chart review at Health Sciences North, a regional stroke centre for northeastern Ontario that currently does not offer endovascular therapy for patients with acute ischemic stroke. Medical records and neurovascular imaging results for patients with a discharge diagnosis of cerebral infarction who were seen by the stroke on-call team at Health Sciences North between May 1, 2016, and Apr. 30, 2017, were retrospectively examined in accordance with criteria of the ESCAPE trial to identify potential candidates for endovascular therapy. The number of candidates was extrapolated to include patients discharged with cerebral infarction from referral centres within a 2-hour transport radius from the study institution. RESULTS: Of 71 patients who met the inclusion criteria, 9 were identified as candidates. Of the 71 cases reviewed, 62 (87%) presented within the window for administration of recombinant tissue plasminogen activator of 4.5 hours from symptom onset, 66 (93%) within the endovascular therapy window of 6 hours and 69 (97%) within the extended endovascular therapy window of 12 hours. Expanding these estimates to include regional referral centres, we conservatively estimated that Health Sciences North has the potential to perform 22 or 23 procedures annually. INTERPRETATION: The projected annual number of procedures met the minimum requirement to maintain competency. Establishing Health Sciences North as a centre for endovascular therapy could be an important step in improving equity in stroke outcomes across northeastern Ontario.

Inactive C-terminal telomerase reverse transcriptase insertion splicing variants are dominant-negative inhibitors of telomerase.

Maintenance of telomere length and structure is essential for cell survival. Telomere synthesis is mediated by the ribonucleoprotein telomerase in 90% of cancer cells, and is regulated mainly by transcription of the human telomerase reverse transcriptase subunit, hTERT. However, transcriptome analysis reveals complex splicing patterns and to date, twenty-two alternatively-spliced hTERT mRNAs have been reported, yet their functions have not been fully elucidated. The best characterized hTERT spliced variants encode for inactive proteins that possess specific deletions within the hTERT catalytic domains. We studied two less well characterized hTERT splice variants (termed INS3 and 4) that encode proteins with intact reverse transcriptase motifs, but alternative C-domains due to insertion of intronic sequences. We determined the prevalence of these mRNA variants in primary cells, telomerase-positive cells and in alternative lengthening of telomere (ALT) cells and found the transcripts to be expressed mainly in telomerase-positive cell lines and to be translated into proteins as illustrated by their association with polysomes. These variants were inactive when expressed in vitro or in cells, retained DNA substrate binding in vitro but were impaired in binding the telomerase RNA component when expressed in, and immunoprecipitated from either telomerase-positive or telomerase-negative ALT cells coexpressing the telomerase RNA component. Stable expression of INS3 and INS4 variants in a hepatocarcinoma cell line inhibited telomerase activity, shortened telomeres and slowed cell growth suggesting a potential dominant-negative function.