scholarly journals Structural features of mouse telomerase RNA are responsible for the lower activity of mouse telomerase versus human telomerase

2006 ◽  
Vol 397 (3) ◽  
pp. 399-406 ◽  
Author(s):  
Scott J. Garforth ◽  
Yan Yun Wu ◽  
Vinayaka R. Prasad
Keyword(s):  
Telomerase Activity ◽  
Structural Features ◽  
In Vitro Translation ◽  
Telomerase Rna ◽  
Level Of Activity ◽  
Human Telomerase ◽  
High Degree ◽  
Made In ◽  
Template Region

Human and mouse telomerases show a high degree of similarity in both the protein and RNA components. Human telomerase is more active and more processive than the mouse telomerase. There are two key differences between hTR [human TR (telomerase RNA)] and mTR (mouse TR) structures. First, the mouse telomerase contains only 2 nt upstream of its template region, whereas the human telomerase contains 45 nt. Secondly, the template region of human telomerase contains a 5-nt alignment domain, whereas that of mouse has only 2 nt. We hypothesize that these differences are responsible for the differential telomerase activities. Mutations were made in both the hTR and mTR, changing the template length and the length of the RNA upstream of the template, and telomerase was reconstituted in vitro using mouse telomerase reverse transcriptase generated by in vitro translation. We show that the sequences upstream of the template region, with a potential to form a double-stranded helix (the P1 helix) as in hTR, increase telomerase activity. The longer alignment domain increases telomerase activity only in the context of the P1 helix. Thus the TR contributes to regulating the level of activity of mammalian telomerases.

scholarly journals Polymerization Defects within Human Telomerase Are Distinct from Telomerase RNA and TEP1 Binding

2000 ◽  
Vol 11 (10) ◽  
pp. 3329-3340 ◽  
Author(s):  
Tara L. Beattie ◽  
Wen Zhou ◽  
Murray O. Robinson ◽  
Lea Harrington
Keyword(s):  
Telomerase Activity ◽  
Amino Terminus ◽  
Telomerase Rna ◽  
Carboxy Terminus ◽  
Active Core ◽  
Human Telomerase ◽  
Precise Role

The minimal, active core of human telomerase is postulated to contain two components, the telomerase RNA hTER and the telomerase reverse transcriptase hTERT. The reconstitution of human telomerase activity in vitro has facilitated the identification of sequences within the telomerase RNA and the RT motifs of hTERT that are essential for telomerase activity. However, the precise role of residues outside the RT domain of hTERT is unknown. Here we have delineated several regions within hTERT that are important for telomerase catalysis, primer use, and interaction with the telomerase RNA and the telomerase-associated protein TEP1. In particular, certain deletions of the amino and carboxy terminus of hTERT that retained an interaction with telomerase RNA and TEP1 were nonetheless completely inactive in vitro and in vivo. Furthermore, hTERT truncations lacking the amino terminus that were competent to bind the telomerase RNA were severely compromised for the ability to elongate telomeric and nontelomeric primers. These results suggest that the interaction of telomerase RNA with hTERT can be functionally uncoupled from polymerization, and that there are regions outside the RT domain of hTERT that are critical for telomerase activity and primer use. These results establish that the human telomerase RT possesses unique polymerization determinants that distinguish it from other RTs.

scholarly journals Physical connectivity mapping by circular permutation of human telomerase RNA reveals new regions critical for activity and processivity

2015 ◽  
pp. MCB.00794-15 ◽  
Author(s):  
Melissa A. Mefford ◽  
David C. Zappulla
Keyword(s):  
Cancer Therapeutics ◽  
Telomerase Activity ◽  
Circular Permutation ◽  
Telomerase Rna ◽  
Recognition Element ◽  
Circular Permutations ◽  
Anti Cancer ◽  
Human Telomerase ◽  
First Time

Telomerase is a specialized ribonucleoprotein complex that extends the 3’ ends of chromosomes to counteract telomere shortening. However, increased telomerase activity is associated with ∼90% of human cancers. The telomerase enzyme minimally requires an RNA (hTR) and a specialized reverse transcriptase protein (TERT) for activityin vitro. Understanding the structure-function relationships within hTR has important implications for human disease. For the first time, we have tested the physical-connectivity requirements in the 451-nucleotide hTR RNA using circular permutations, which reposition the 5’ and 3’ ends. Our extensivein vitroanalysis identified three classes of hTR circular permutants with altered function. First, circularly permuting 3’ of the template causes specific defects in repeat-addition processivity, revealing that the template-recognition element found in ciliates is conserved in human telomerase RNA. Second, seven circular permutations residing within the catalytically important core and CR4/5 domains completely abolish telomerase activity, unveiling mechanistically critical portions of these domains. Third, several circular permutations between the core and CR4/5 significantly increase telomerase activity. Our extensive circular permutation results provide insights into the architecture and coordination of human hTR and highlight where the RNA could be targeted for the development of anti-aging and anti-cancer therapeutics.

scholarly journals The Product of the Survival of Motor Neuron(SMN) Gene is a Human Telomerase-associated Protein

2002 ◽  
Vol 13 (9) ◽  
pp. 3192-3202 ◽  
Author(s):  
François Bachand ◽  
François-Michel Boisvert ◽  
Jocelyn Côté ◽  
Stéphane Richard ◽  
Chantal Autexier
Keyword(s):  
Motor Neuron ◽  
Telomerase Activity ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Telomerase Rna ◽  
Sm Proteins ◽  
Rnp Complex ◽  
Smn Gene ◽  
Human Telomerase

Telomerase is a ribonucleoprotein (RNP) complex that is minimally composed of a protein catalytic subunit, the telomerase reverse transcriptase (TERT), and an RNA component, the telomerase RNA. Thesurvival of motor neuron (SMN) gene codes for a protein involved in the biogenesis of certain RNPs. Here, we report that SMN is a telomerase-associated protein. Using in vitro binding assays and immunoprecipitation experiments, we demonstrate an association between SMN and the telomerase RNP in vitro and in human cells. The specific immunopurification of SMN from human 293 cells copurified telomerase activity, suggesting that SMN associates with a subset of the functional telomerase holoenzyme. Our results also indicate that the human telomerase RNA and the human (h) TERT are not associated with Sm proteins, in contrast to Saccharomyces cerevisiae telomerase. Immunofluorescence analysis showed that hTERT does not specifically colocalize with wild-type SMN in gems or Cajal bodies. However, a dominant-negative mutant of SMN (SMNΔN27) previously characterized to elicit the cellular reorganization of small nuclear RNPs caused the accumulation of hTERT in specific SMNΔN27-induced cellular bodies. Furthermore, coexpression of SMNΔN27 and hTERT in rabbit reticulocyte lysates decreased the efficiency of human telomerase reconstitution in vitro. Our results establish SMN as a novel telomerase-associated protein that is likely to function in human telomerase biogenesis.

scholarly journals Peculiarities of Yeasts and Human Telomerase RNAs Processing

Acta Naturae ◽  
2016 ◽  
Vol 8 (4) ◽  
pp. 14-22 ◽  
Author(s):  
M. P. Rubtsova ◽  
D. P. Vasilkova ◽  
Yu. V. Naraykina ◽  
O. A. Dontsova
Keyword(s):  
Reverse Transcriptase ◽  
Somatic Cells ◽  
Telomerase Activity ◽  
Proliferative Potential ◽  
Telomerase Rna ◽  
Embryonic Cells ◽  
Eukaryotic Chromosome ◽  
Human Telomerase ◽  
Linear Chromosomes

Telomerase is one of the major components of the telomeres -- linear eukaryotic chromosome ends - maintenance system. Linear chromosomes are shortened during each cell division due to the removal of the primer used for DNA replication. Special repeated telomere sequences at the very ends of linear chromosomes prevent the deletion of genome information caused by primer removal. Telomeres are shortened at each replication round until it becomes critically short and is no longer able to protect the chromosome in somatic cells. At this stage, a cell undergoes a crisis and usually dies. Rare cases result in telomerase activation, and the cell gains unlimited proliferative capacity. Special types of cells, such as stem, germ, embryonic cells and cells from tissues with a high proliferative potential, maintain their telomerase activity indefinitely. The telomerase is inactive in the majority of somatic cells. Telomerase activity in vitro requires two key components: telomerase reverse transcriptase and telomerase RNA. In cancer cells, telomerase reactivates due to the expression of the reverse transcriptase gene. Telomerase RNA expresses constitutively in the majority of human cells. This fact suggests that there are alternative functions to telomerase RNA that are unknown at the moment. In this manuscript, we review the biogenesis of yeasts and human telomerase RNAs thanks to breakthroughs achieved in research on telomerase RNA processing by different yeasts species and humans in the last several years.

scholarly journals The human telomerase catalytic subunit and viral telomerase RNA reconstitute a functional telomerase complex in a cell-free system, but not in human cells

2012 ◽  
Vol 17 (4) ◽  
Author(s):  
Laetitia Trapp-Fragnet ◽  
Delphine Marie-Egyptienne ◽  
Johans Fakhoury ◽  
Denis Rasschaert ◽  
Chantal Autexier
Keyword(s):  
Telomerase Activity ◽  
Human Cells ◽  
Telomerase Rna ◽  
Free System ◽  
Cell Free System ◽  
Associated Proteins ◽  
A Cell ◽  
Human Telomerase ◽  
Free Environment

AbstractThe minimal vertebrate telomerase enzyme is composed of a protein component (telomerase reverse transcriptase, TERT) and an RNA component (telomerase RNA, TR). Expression of these two subunits is sufficient to reconstitute telomerase activity in vitro, while the formation of a holoenzyme comprising telomerase-associated proteins is necessary for proper telomere length maintenance. Previous reports demonstrated the high processivity of the human telomerase complex and the interspecies compatibility of human TERT (hTERT). In this study, we tested the function of the only known viral telomerase RNA subunit (vTR) in association with human telomerase, both in a cell-free system and in human cells. When vTR is assembled with hTERT in a cell-free environment, it is able to interact with hTERT and to reconstitute telomerase activity. However, in human cells, vTR does not reconstitute telomerase activity and could not be detected in the human telomerase complex, suggesting that vTR is not able to interact properly with the proteins constituting the human telomerase holoenzyme.

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 Functional Multimerization of the Human Telomerase Reverse Transcriptase

2001 ◽  
Vol 21 (18) ◽  
pp. 6151-6160 ◽  
Author(s):  
Tara L. Beattie ◽  
Wen Zhou ◽  
Murray O. Robinson ◽  
Lea Harrington
Keyword(s):  
Reverse Transcriptase ◽  
Telomerase Activity ◽  
Telomerase Reverse Transcriptase ◽  
Telomerase Rna ◽  
Human Telomerase Reverse Transcriptase ◽  
Catalytic Core ◽  
Cell Extracts ◽  
Human Telomerase

ABSTRACT The telomerase enzyme exists as a large complex (∼1,000 kDa) in mammals and at minimum is composed of the telomerase RNA and the catalytic subunit telomerase reverse transcriptase (TERT). In Saccharomyces cerevisiae, telomerase appears to function as an interdependent dimer or multimer in vivo (J. Prescott and E. H. Blackburn, Genes Dev. 11:2790–2800, 1997). However, the requirements for multimerization are not known, and it remained unclear whether telomerase exists as a multimer in other organisms. We show here that human TERT (hTERT) forms a functional multimer in a rabbit reticulocyte lysate reconstitution assay and in human cell extracts. Two separate, catalytically inactive TERT proteins can complement each other in trans to reconstitute catalytic activity. This complementation requires the amino terminus of one hTERT and the reverse transcriptase and C-terminal domains of the second hTERT. The telomerase RNA must associate with only the latter hTERT for reconstitution of telomerase activity to occur. Multimerization of telomerase also facilitates the recognition and elongation of substrates in vitro and in vivo. These data suggest that the catalytic core of human telomerase may exist as a functionally cooperative dimer or multimer in vivo.

scholarly journals Stem-Loop IV of Tetrahymena Telomerase RNA Stimulates Processivity in trans

2003 ◽  
Vol 23 (16) ◽  
pp. 5606-5613 ◽  
Author(s):  
Douglas X. Mason ◽  
Elizabeth Goneska ◽  
Carol W. Greider
Keyword(s):  
Telomerase Activity ◽  
Telomerase Rna ◽  
In Vitro Activity ◽  
Wild Type ◽  
Protein Subunit ◽  
Stem Loop ◽  
Cell Growth Arrest ◽  
In Trans ◽  
Template Region

ABSTRACT Telomerase is a ribonucleoprotein enzyme responsible for the addition of telomeres onto the ends of chromosomes. Short or dysfunctional telomeres can lead to cell growth arrest, apoptosis, and genomic instability. Telomerase uses its RNA subunit to copy a short template region for telomere synthesis. To probe for regions of Tetrahymena telomerase RNA essential for function, we assayed 27 circularly permuted RNA deletions for telomerase in vitro activity and binding to the telomerase reverse transcriptase catalytic protein subunit. We found that stem-loop IV is required for wild-type telomerase activity in vitro and will stimulate processivity when added in trans.

Structural and Genetic Aspects of Proline-rich Proteins

1987 ◽  
Vol 66 (2) ◽  
pp. 457-461 ◽  
Author(s):  
A. Bennick
Keyword(s):  
General Structure ◽  
Single Gene ◽  
Conclusive Evidence ◽  
In Vitro Translation ◽  
Binding Studies ◽  
Nmr Studies ◽  
Post Translational Modifications ◽  
Single Precursor ◽  
Made In

Considerable advances have been made in the genetics of salivary proline-rich proteins (PRP). The genes for acidic, basic, and glycosylated PRP have been cloned. They code for precursor proteins that all have an acidic N-terminal followed by proline-rich repeat sequences. Structural studies on secreted proteins have demonstrated that not only acidic but also some basic PRPs have this general structure. It is possible that mRNA for different PRP may have originated from a single gene by differential mRNA splicing, but post-translational cleavages of the primary translation product apparently also occur. In vitro translation of salivary gland mRNA results in a single precursor protein for acidic PRP. Such in vitro translated protein can be cleaved by salivary kallikrein, giving rise to two commonly secreted acidic PRPs, and kallikrein or kallikrein-like enzymes may be responsible for other post-translational cleavages of PRPs. Acidic as well as some basic PRPs are phosphorylated. A protein kinase has been demonstrated in salivary glands which phosphorylates the PRPs and other secreted salivary proteins in a cAMP and Ca2+-calmodulinindependent manner. Knowledge of the conformation of PRPs is limited. There is no conclusive evidence of polyproline-like structure in the proline-rich part of PRPs. Ca2+ binding studies on acidic PRPs indicate that there is interaction between the Ca2+ binding N-terminal end and the proline-rich C-terminal part. This interaction is relieved by modification of arginine side-chains. 1H, 32P, and 43Ca NMR studies have further elucidated the conformation of acidic PRPs in solution. Present evidence shows that salivary PRPs constitute a unique superfamily of proteins which pose a number of interesting questions concerning gene structure, pre- and post-translational modifications, and protein conformation.

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