scholarly journals Functional Multimerization of Human Telomerase Requires an RNA Interaction Domain in the N Terminus of the Catalytic Subunit

2002 ◽  
Vol 22 (4) ◽  
pp. 1253-1265 ◽  
Author(s):  
Tara J. Moriarty ◽  
Sylvain Huard ◽  
Sophie Dupuis ◽  
Chantal Autexier
Keyword(s):  
Reverse Transcriptase ◽  
Rna Binding ◽  
Catalytic Subunit ◽  
Binding Motif ◽  
Telomerase Rna ◽  
Human Telomerase Reverse Transcriptase ◽  
Interaction Domain ◽  
N Terminus ◽  
Human Telomerase ◽  
Rna Interaction

ABSTRACT Functional human telomerase complexes are minimally composed of the human telomerase RNA (hTR) and a catalytic subunit (human telomerase reverse transcriptase [hTERT]) containing reverse transcriptase (RT)-like motifs. The N terminus of TERT proteins is unique to the telomerase family and has been implicated in catalysis, telomerase RNA binding, and telomerase multimerization, and conserved motifs have been identified by alignment of TERT sequences from multiple organisms. We studied hTERT proteins containing N-terminal deletions or substitutions to identify and characterize hTERT domains mediating telomerase catalytic activity, hTR binding, and hTERT multimerization. Using multiple sequence alignment, we identified two vertebrate-conserved TERT N-terminal regions containing vertebrate-specific residues that were required for human telomerase activity. We identified two RNA interaction domains, RID1 and RID2, the latter containing a vertebrate-specific RNA binding motif. Mutations in RID2 reduced the association of hTR with hTERT by 50 to 70%. Inactive mutants defective in RID2-mediated hTR binding failed to complement an inactive hTERT mutant containing an RT motif substitution to reconstitute activity. Our results suggest that functional hTERT complementation requires intact RID2 and RT domains on the same hTERT molecule and is dependent on hTR and the N terminus.

scholarly journals Functional Regions of Human Telomerase Reverse Transcriptase and Human Telomerase RNA Required for Telomerase Activity and RNA-Protein Interactions

2001 ◽  
Vol 21 (5) ◽  
pp. 1888-1897 ◽  
Author(s):  
François Bachand ◽  
Chantal Autexier
Keyword(s):  
Reverse Transcriptase ◽  
Rna Binding ◽  
Telomerase Activity ◽  
Catalytic Subunit ◽  
Telomerase Rna ◽  
Dna Repeats ◽  
Human Telomerase Reverse Transcriptase ◽  
Template Sequence ◽  
Human Telomerase

ABSTRACT Telomerase is a specialized reverse transcriptase (RT) that is minimally composed of a protein catalytic subunit and an RNA component. The RNA subunit contains a short template sequence that directs the synthesis of DNA repeats at the ends of chromosomes. Human telomerase activity can be reconstituted in vitro by the expression of the human telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. We analyzed telomerase activity and binding of hTR to hTERT in RRL by expressing different hTERT and hTR variants. hTRs containing nucleotide substitutions that are predicted to disrupt base pairing in the P3 helix of the pseudoknot weakly reconstituted human telomerase activity yet retained their ability to bind hTERT. Our results also identified two distinct regions of hTR that can independently bind hTERT in vitro. Furthermore, sequences or structures between nucleotides 208 and 330 of hTR (which include the conserved CR4-CR5 domain) were found to be important for hTERT-hTR interactions and for telomerase activity reconstitution. Human TERT carboxy-terminal amino acid deletions extending to motif E or the deletion of the first 280 amino acids abolished human telomerase activity without affecting the ability of hTERT to associate with hTR, suggesting that the RT and RNA binding functions of hTERT are separable. These results indicate that the reconstitution of human telomerase activity in vitro requires regions of hTERT that (i) are distinct from the conserved RT motifs and (ii) bind nucleotides distal to the hTR template sequence.

scholarly journals Telomerase Activity Reconstitutedin Vitrowith Purified Human Telomerase Reverse Transcriptase and Human Telomerase RNA Component

2000 ◽  
Vol 275 (29) ◽  
pp. 22568-22573 ◽  
Author(s):  
Kenkichi Masutomi ◽  
Shuichi Kaneko ◽  
Naoyuki Hayashi ◽  
Tatsuya Yamashita ◽  
Yukihiro Shirota ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Telomerase Activity ◽  
Telomerase Reverse Transcriptase ◽  
Telomerase Rna ◽  
Human Telomerase Reverse Transcriptase ◽  
Human Telomerase

scholarly journals Usefulness of Immunological Detection of the Human Telomerase Reverse Transcriptase

2005 ◽  
Vol 27 (5-6) ◽  
pp. 347-353
Author(s):  
Annalisa Volpi ◽  
Sara Bravaccini ◽  
Laura Medri ◽  
Serenella Cerasoli ◽  
Michele Gaudio ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Reverse Transcriptase ◽  
Precancerous Lesions ◽  
Pyramidal Cells ◽  
Catalytic Subunit ◽  
Telomerase Reverse Transcriptase ◽  
Human Telomerase Reverse Transcriptase ◽  
Enzymatic Function ◽  
Somatic Tissues ◽  
Human Telomerase

Objective: Recent years have seen a considerable wealth of studies conducted on the potential usefulness of telomerase determination in diagnosis, prognosis and targeted cancer therapy. The frequently used Telomeric Repeat Amplification Protocol assay suffers from some drawbacks, the most important being the rate of false positives. In situ analysis using well characterised antibodies directed against the human telomerase reverse transcriptase (hTERT) would therefore appear to be important to morphologically identify the nature of telomerase positive cells. Methods: We performed immunostaining in a series of cultured cells and in normal, preneoplastic and tumour tissues from different organs using a monoclonal antibody directed against the catalytic subunit of telomerase. Results: Immunoreactivity was not observed in perennial cells of terminally differentiated cardiac and skeletal muscular tissues or in small pyramidal cells of the cerebral cortex. Conversely, it was found in other normal somatic tissues as well as in precancerous lesions and in all tumour histotypes. Conclusions: Immunohistochemistry with a well characterised hTERT-specific monoclonal antibody permitted the identification of hTERT immunopositive cells in normal somatic tissues. Whether hTERT protein detected by immunostaining with hTERT-specific Tel 3 36-10 antibody is actually the degraded form of the protein that retains hTERT antigenicity but not enzymatic function, or whether it represents the real, potentially functional catalytic subunit of the enzyme, immunohistochemistry would not seem to represent a useful tool to investigate the role of telomerase and the mechanisms involved in its regulation.

scholarly journals RNA Binding Domain of Telomerase Reverse Transcriptase

2001 ◽  
Vol 21 (4) ◽  
pp. 990-1000 ◽  
Author(s):  
Cary K. Lai ◽  
James R. Mitchell ◽  
Kathleen Collins
Keyword(s):  
Catalytic Activity ◽  
Reverse Transcriptase ◽  
Rna Binding ◽  
Binding Domain ◽  
Amino Terminus ◽  
Telomerase Reverse Transcriptase ◽  
Telomerase Rna ◽  
Sequence Motifs ◽  
Rna Binding Domain ◽  
Rna Interaction

ABSTRACT Telomerase is a ribonucleoprotein reverse transcriptase that extends the ends of chromosomes. The two telomerase subunits essential for catalysis in vitro are the telomerase reverse transcriptase (TERT) and the telomerase RNA. Using truncations and site-specific mutations, we identified sequence elements of TERT and telomerase RNA required for catalytic activity and protein-RNA interaction for Tetrahymena thermophila telomerase. We found that the TERT amino and carboxyl termini, although evolutionarily poorly conserved, are nonetheless important for catalytic activity. In contrast, high-affinity telomerase RNA binding requires only a small region in the amino terminus of TERT. Surprisingly, the TERT region necessary and sufficient for telomerase RNA binding is completely separable from the reverse transcriptase motifs. The minimalTetrahymena TERT RNA binding domain contains two sequence motifs with ciliate-specific conservation and one TERT motif with conservation across all species. With human TERT, we demonstrate that a similar region within the TERT amino terminus is essential for human telomerase RNA binding as well. Finally, we defined theTetrahymena telomerase RNA sequences that are essential for TERT interaction. We found that a four-nucleotide region 5′ of the template is critical for TERT binding and that the 5′ end of telomerase RNA is sufficient for TERT binding. Our results reveal at least one evolutionarily conserved molecular mechanism by which the telomerase reverse transcriptase is functionally specialized for obligate use of an internal RNA template.

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