title: An intact putative mouse telomerase essential N-terminal domain is necessary for proper telomere maintenance.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec, H3T 1E2, Canada.
authors.forename: Philippe
authors.initials: P
authors.lastname: Rousseau
authors.affiliation: Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec, H3T 1E2, Canada.|Division of Experimental Medicine, McGill University, Montréal, Québec, Canada.
authors.forename: Shanjadia
authors.initials: S
authors.lastname: Khondaker
authors.affiliation: Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec, H3T 1E2, Canada.
authors.forename: Shusen
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec, H3T 1E2, Canada.
authors.forename: Catherine
authors.initials: C
authors.lastname: Lauzon
authors.affiliation: Manitoba Institute of Cell Biology, University of Manitoba, Manitoba, R3E 0V9, Canada.
authors.forename: Sabine
authors.initials: S
authors.lastname: Mai
authors.affiliation: Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec, H3T 1E2, Canada.|Division of Experimental Medicine, McGill University, Montréal, Québec, Canada.|Department of Anatomy and Cell Biology, McGill University, Montréal, Québec, Canada.
authors.forename: Chantal
authors.initials: C
authors.lastname: Autexier
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D051379:Mice; D014176:Protein Biosynthesis; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D019098:Telomerase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D020033:Protein Isoforms; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase; C509187:Tert protein, mouse
keywords: Alternative splicing; Dominant-negative; Isoform; Telomerase; Telomeres