Telomerase is an enzyme that maintains telomere length by adding telomeric sequence repeats onto chromosome ends. The telomerase ribonucleoprotein complex consists of two essential components, a reverse transcriptase and an RNA molecule that provides the template for telomeric repeat synthesis. A common secondary structure of vertebrate telomerase RNA has been proposed based on a phylogenetic comparative analysis of 35 sequences. Here we report the identification of an additional essential base-paired region in the CR4-CR5 domain of mammalian telomerase RNA, termed P6.1. Mouse telomerase RNAs with mutations that disrupted base pairings in the P6.1 helix were unable to reconstitute telomerase activity in vivo. In contrast, an RNA mutant with compensatory mutations that restored base pairings in the P6.1 helix restored telomerase activity. In an in vitro reconstitution system stable base pairing of the P6.1 stem was required for the RNA-protein interaction between the CR4-CR5 domain and the telomerase reverse transcriptase (TERT) protein. Interestingly, two RNA mutations, one that extends the P6.1 stem and one that alters the conserved nucleotides of the L6.1 loop, allowed RNA-protein binding but significantly impaired telomerase activity. These data establish the presence of the P6.1 stem-loop and its importance for the assembly and enzymatic activity of the mammalian telomerase complex.
Telomeres are usually maintained about an equilibrium length, and the set point for this equilibrium differs between species and between strains of a given species. To examine the requirement for telomerase in mediating establishment of a new telomere length equilibrium, we generated interspecies crosses with telomerase mTR knockout mice. In crosses between C57BL͞6J (B6) and either of two unrelated mouse species, CAST͞Ei and SPRET͞Ei, telomerase mediated establishment of a new telomere length equilibrium in wild-type mTR ؉/؉ mice. This new equilibrium was characterized by elongation of the short telomeres of CAST͞Ei or SPRET͞Ei origin. In contrast, mTR ؊/؊ offspring of interspecies crosses failed to elongate telomeres. Unexpectedly, haploinsufficiency was observed in mTR ؉/؊ heterozygous interspecies mice, which had an impaired ability to elongate short SPRET͞Ei or CAST͞Ei telomeres to the new equilibrium set point that was achieved in wild-type mTR ؉/؉ mice. These results demonstrate that elongation of telomeres to a new telomere set point requires telomerase and indicate that telomerase RNA may be limiting in vivo.
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