Reconstitution of human telomerase activity and identification of a minimal functional region of the human telomerase RNA.

Telomerase is a ribonucleoprotein that catalyzes telomere elongation through the addition of TTAGGG repeats in humans. Activation of telomerase is often associated with immortalization of human cells and cancer. To dissect the human telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential 445 nucleotide human telomerase RNA (hTR) by micrococcal nuclease digestion of partially purified human telomerase, the addition of in vitro transcribed hTR reconstituted telomerase activity. The activity was dependent upon and specific to hTR. Using this assay, truncations at the 5′ and 3′ ends of hTR identified a functional region of hTR, similar in size to the full‐length telomerase RNAs from ciliates. This region is located between positions 1‐203. Furthermore, we found that residues 1‐44, 5′ to the template region (residues 46–56) are not essential for activity, indicating a minimal functional region is located between residues 44–203. Mutagenesis of full‐length hTR between residues 170–179, 180–189 or 190–199 almost completely abolished the ability of the hTR to function in the reconstitution of telomerase activity, suggesting that sequences or structures within this 30 nucleotide region are required for activity, perhaps by binding telomerase protein components.

[1]  D. Romero,et al.  A single telomerase RNA is sufficient for the synthesis of variable telomeric DNA repeats in ciliates of the genus Paramecium , 1996, Molecular and cellular biology.

[2]  T. Cech,et al.  Method for determining RNA 3' ends and application to human telomerase RNA. , 1996, Nucleic acids research.

[3]  S. Bacchetti.  Telomere dynamics and telomerase activity in cell senescence and cancer , 1996 .

[4]  C. Autexier,et al.  Boundary elements of the Tetrahymena telomerase RNA template and alignment domains. , 1995, Genes & development.

[5]  S. Bacchetti,et al.  Telomeres and telomerase in human cancer (review). , 1995, International journal of oncology.

[6]  M. Blasco,et al.  Functional characterization and developmental regulation of mouse telomerase RNA , 1995, Science.

[7]  E. Blackburn,et al.  Runaway telomere elongation caused by telomerase RNA gene mutations , 1995, Nature.

[8]  B. Sproat,et al.  In vitro reconstitution of mammalian U2 and U5 snRNPs active in splicing: Sm proteins are functionally interchangeable and are essential for the formation of functional U2 and U5 snRNPs. , 1995, The EMBO journal.

[9]  J. Shay,et al.  Time, telomeres and tumours: is cellular senescence more than an anticancer mechanism? , 1995, Trends in cell biology.

[10]  R. Kobayashi,et al.  Purification of tetrahymena telomerase and cloning of genes encoding the two protein components of the enzyme , 1995, Cell.

[11]  C B Harley,et al.  Specific association of human telomerase activity with immortal cells and cancer. , 1994, Science.

[12]  T. Cech,et al.  Telomerase RNAs of different ciliates have a common secondary structure and a permuted template. , 1994, Genes & development.

[13]  C. Autexier,et al.  Functional reconstitution of wild-type and mutant Tetrahymena telomerase. , 1994, Genes & development.

[14]  C B Harley,et al.  Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes. , 1993, American journal of human genetics.

[15]  C B Harley,et al.  Telomere length predicts replicative capacity of human fibroblasts. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[16]  J. Steitz,et al.  Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. , 1992, The EMBO journal.

[17]  C. Guthrie,et al.  More than half of yeast U1 snRNA is dispensable for growth. , 1991, Nucleic acids research.

[18]  Daniel P. Romero,et al.  A conserved secondary structure for telomerase RNA , 1991, Cell.

[19]  P. Walter,et al.  Binding sites of the 9- and 14-kilodalton heterodimeric protein subunit of the signal recognition particle (SRP) are contained exclusively in the Alu domain of SRP RNA and contain a sequence motif that is conserved in evolution. , 1991, Molecular and cellular biology.

[20]  K. Nierhaus,et al.  The assembly of prokaryotic ribosomes. , 1991, Biochimie.

[21]  Robin C. Allshire,et al.  Telomere reduction in human colorectal carcinoma and with ageing , 1990, Nature.

[22]  C. Harley,et al.  Telomeres shorten during ageing of human fibroblasts , 1990, Nature.

[23]  E. Blackburn,et al.  In vivo alteration of telomere sequences and senescence caused by mutated Tetrahymena telomerase RNAs , 1990, Nature.

[24]  R. Myers,et al.  Structure and variability of human chromosome ends , 1990, Molecular and cellular biology.

[25]  N. Pace,et al.  The design and catalytic properties of a simplified ribonuclease P RNA. , 1989, Science.

[26]  E. Blackburn,et al.  A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis , 1989, Nature.

[27]  C. Guthrie,et al.  Two conserved domains of yeast U2 snRNA are separated by 945 nonessential nucleotides , 1988, Cell.

[28]  M. Ares,et al.  Internal sequences that distinguish yeast from metazoan U2 snRNA are unnecessary for pre-mRNA splicing , 1988, Nature.

[29]  M. Ares.  U2 RNA from yeast is unexpectedly large and contains homology to vertebrate U4, U5, and U6 small nuclear RNAs , 1986, Cell.

[30]  Carol W. Greider,et al.  Identification of a specific telomere terminal transferase activity in tetrahymena extracts , 1985, Cell.

[31]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[32]  C. Greider.  Telomere length regulation. , 1996, Annual review of biochemistry.

[33]  C. Harley,et al.  Telomerase, cell immortality, and cancer. , 1994, Cold Spring Harbor symposia on quantitative biology.

[34]  H. Cooke,et al.  In vivo loss of telomeric repeats with age in humans. , 1991, Mutation research.