J. Lingner - Academia.edu (original) (raw)

Papers by J. Lingner

Oncogene, 2001

Telomerase activation is crucial in human carcinogenesis. The limiting component of telomerase, t... more Telomerase activation is crucial in human carcinogenesis. The limiting component of telomerase, the catalytic subunit (hTERT), is undetectable in normal somatic cells but present in most tumor cells, including the earliest stages of colon carcinoma. The mechanisms involved in the dierential expression in normal and tumor cells are not understood. In normal cells hTERT expression is shut down by a repressor, and upregulation could be a consequence of cis-acting changes in the hTERT gene, making it resistant to repression. We have identi®ed a polymorphic and a monomorphic minisatellite in the second intron of the hTERT gene, and polymorphic one in intron 6. The polymorphic minisatellite in intron 2 contains binding sites for c-Myc, which has been shown to upregulate hTERT transcription. Screening colon carcinoma DNAs for rearrangements of hTERT minisatellites we detected no changes in 33 samples from tumors, most of which express hTERT. This indicates that size rearrangements of the hTERT minisatellites are not required for telomerase expression in colon carcinomas. Minor changes and one LOH were seen in ®ve tumors.

Nature genetics, 1999

The MYC proto-oncogene encodes a ubiquitous transcription factor (c-MYC) involved in the control ... more The MYC proto-oncogene encodes a ubiquitous transcription factor (c-MYC) involved in the control of cell proliferation and differentiation. Deregulated expression of c-MYC caused by gene amplification, retroviral insertion, or chromosomal translocation is associated with tumorigenesis. The function of c-MYC and its role in tumorigenesis are poorly understood because few c-MYC targets have been identified. Here we show that c-MYC has a direct role in induction of the activity of telomerase, the ribonucleoprotein complex expressed in proliferating and transformed cells, in which it preserves chromosome integrity by maintaining telomere length. c-MYC activates telomerase by inducing expression of its catalytic subunit, telomerase reverse transcriptase (TERT). Telomerase complex activity is dependent on TERT, a specialized type of reverse transcriptase. TERT and c-MYC are expressed in normal and transformed proliferating cells, downregulated in quiescent and terminally differentiated ce...

Ciba Foundation symposium, 1997

Telomerase, the enzyme that extends chromosomal DNA ends in most eukaryotes, contains essential R... more Telomerase, the enzyme that extends chromosomal DNA ends in most eukaryotes, contains essential RNA and protein subunits. We have been studying telomere replication in hypotrichous ciliates such as Euplotes aediculatus, which have numerous short macronuclear DNA molecules and therefore are highly enriched in telomeres and in telomerase. Cloning and sequencing genes for the RNA subunits from several ciliates revealed that telomerase RNAs with insignificant nucleotide sequence homology nevertheless form a common secondary structure. Affinity chromatography based on the sequence of the RNA subunit was used to purify the Euplotes telomerase as an active ribonucleoprotein enzyme. Two protein subunits, 123 kDa and 43 kDa, were identified. The finding of a yeast homologue to the 123 kDa subunit suggests that telomerase protein components may be much more highly conserved in evolution than the RNA subunits. The purified Euplotes telomerase has no activity with blunt-ended DNA primers, but i...

Biochemistry. Biokhimii͡a, 1997

Synthesis of telomeric repeats at chromosome ends requires telomerase, a ribonucleoprotein enzyme... more Synthesis of telomeric repeats at chromosome ends requires telomerase, a ribonucleoprotein enzyme. The RNA subunit, which contains the template for DNA synthesis, has been identified in many organisms. Recently, the protein subunit that catalyzes telomeric DNA extension has also been identified in Euplotes aediculatus and Saccharomyces cerevisiae. It has sequence and functional characteristics of a reverse transcriptase related to retrotransposon and retroviral reverse transcriptases, so this new family of telomerase subunits has been named TRT (Telomerase Reverse Transcriptase). We find it remarkable that the same type of protein structure required for retroviral replication is now seen to be essential for normal chromosome telomere replication in diverse eukaryotes.

Antisense oligonucleotides have been used to study the structure and function of small nuclear ri... more Antisense oligonucleotides have been used to study the structure and function of small nuclear ribonucleoprotein (snRNP) complexes and were adapted and modified for the purification of a variety of RNPs. We describe methods for recombinant expression and reconstitution of catalytically active human telomerase and the purification of native and recombinant telomerase using antisense affinity oligonucleotides. The purification procedure involves binding of the RNP complex to NeutrAvidin beads via a biotinylated 2'-O-methyl (2'-OMe) RNA oligonucleotide complementary to the RNA subunit. The complex is eluted from the beads through competition with a displacement oligonucleotide. Thus, the purified RNP is eluted under mild conditions, retaining its catalytic activity.

The EMBO Journal, 2000

Telomerase is the ribonucleoprotein enzyme responsible for the replication of chromosome ends in ... more Telomerase is the ribonucleoprotein enzyme responsible for the replication of chromosome ends in most eukaryotes. In the ciliate Euplotes aediculatus, the protein p43 biochemically co-puri®es with active telomerase and appears to be stoichiometric with both the RNA and the catalytic protein subunit of this telomerase complex. Here we describe cloning of the gene for p43 and present evidence that it is an authentic component of the telomerase holoenzyme. Comparison of the nucleotide sequence of the cloned gene with peptide sequences of the protein suggests that production of full-length p43 relies on a programmed ribosomal frameshift, an extremely rare translational mechanism. Anti-p43 antibodies immunodeplete telomerase RNA and telomerase activity from E.aediculatus nuclear extracts, indicating that the vast majority of mature telomerase complexes in the cell are associated with p43. The sequence of p43 reveals similarity to the La autoantigen, an RNAbinding protein involved in maturation of RNA polymerase III transcripts, and recombinant p43 binds telomerase RNA in vitro. By analogy to other La proteins, p43 may function in chaperoning the assembly and/or facilitating nuclear retention of telomerase.

Science, 2008

clicking here. colleagues, clients, or customers by , you can order high-quality copies for your ... more clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles

Science, 1995

... SCIENCE * VOL. 269 * 15 SEPTEMBER 1995 Telomerase and DNA End Replication: No Longer a Laggin... more ... SCIENCE * VOL. 269 * 15 SEPTEMBER 1995 Telomerase and DNA End Replication: No Longer a Lagging Strand Problem? Joachim Lingner, Julia Promisel Cooper, Thomas R. Cech 5' ....3' Tetomerase extends =overhang 3' 59 3rtSA

Proceedings of the National Academy of Sciences, 1996

Telomerase is a ribonucleoprotein enzyme that uses its internal RNA moiety as a template for synt... more Telomerase is a ribonucleoprotein enzyme that uses its internal RNA moiety as a template for synthesis of telomeric repeats at chromosome ends. Here we report the purification of'telomerase from Euplotes aediculatus by affinity chromatography with antisense 2'-O-methyl oligonucleotides, a method that was developed for small nuclear ribonucleoprotein particles (snRNPs). Elution of bound ribonucleoprotein from the antisense oligonucleotide under nondenaturing conditions was achieved by a novel approach, using a displacement oligonucleotide. Polypeptides of 12Q kDa and 43 kDa (a doublet) copurify with the active telomerase and appear stoichiometric with telomerase RNA. A simple model for DNA end replication predicts that after semiconservative DNA replication, telomerase will extend the newly synthesized, blunt-ended leading strand. We show that purified Euplotes telomerase has no activity with blunt-ended primers. Instead, efficient extension requires 4 to 6 single-stranded nucleotides at the 3' end. Therefore, this model predicts the existence of other activities such as helicases or nucleases that generate a single-stranded 3' end from a blunt end, thus'activating the end for telomerase extension.

Nucleic Acids Research, 1993

Two commonly used methods to end-label RNAmolecules are 5'-end labeling by polynucleotlde klnase ... more Two commonly used methods to end-label RNAmolecules are 5'-end labeling by polynucleotlde klnase and 3'-end labeling with pCp and T4 RNA llgase. We show here that RNA 3'-ends can also be labeled with the chain-terminating analogue cordycepln 5'-trlphosphate (3'-deoxy-ATP) which Is added by poly(A) polymerase. For a synthetic RNA It Is shown that 40% of cordycepin becomes Incorporated when the nucleotide Is used at limiting concentrations and that with an excess of cordycepin 5'-triphosphate essentially all the RNA becomes modified at Its 3'-end. The reaction Is complete within minutes and the RNA product is uniform and suitable for sequence analysis. The efficiency of labeling varies with different RNAmolecules and is different from RNA ligase. Poly(A) polymerase preferentially labels longer RNA-molecules whereas short RNA-molecules are labeled more efficiently by T4 RNA llgase.

Nucleic Acids Research, 2010

Telomeres, the physical ends of eukaryotes chromosomes are transcribed into telomeric repeat cont... more Telomeres, the physical ends of eukaryotes chromosomes are transcribed into telomeric repeat containing RNA (TERRA), a large non-coding RNA of unknown function, which forms an integral part of telomeric heterochromatin. TERRA molecules resemble in sequence the telomeric DNA substrate as they contain 5 0-UUAGGG-3 0 repeats near their 3 0-end which are complementary to the template sequence of telomerase RNA. Here we demonstrate that endogenous TERRA is bound to human telomerase in cell extracts. Using in vitro reconstituted telomerase and synthetic TERRA molecules we demonstrate that the 5 0-UUAGGG-3 0 repeats of TERRA base pair with the RNA template of the telomerase RNA moiety (TR). In addition TERRA contacts the telomerase reverse transcriptase (TERT) protein subunit independently of hTR. In vitro studies further demonstrate that TERRA is not used as a telomerase substrate. Instead, TERRA acts as a potent competitive inhibitor for telomeric DNA in addition to exerting an uncompetitive mode of inhibition. Our data identify TERRA as a telomerase ligand and natural direct inhibitor of human telomerase. Telomerase regulation by the telomere substrate may be mediated via its transcription.

Nucleic Acids Research, 2007

The Smg proteins Smg5, Smg6 and Smg7 are involved in nonsense-mediated RNA decay (NMD) in metazoa... more The Smg proteins Smg5, Smg6 and Smg7 are involved in nonsense-mediated RNA decay (NMD) in metazoans, but no orthologs have been found in the budding yeast Saccharomyces cerevisiae. Sequence alignments reveal that yeast Ebs1p is similar in structure to the human Smg5-7, with highest homology to Smg7. We demonstrate here that Ebs1p is involved in NMD and behaves similarly to human Smg proteins. Indeed, both loss and overexpression of Ebs1p results in stabilization of NMD targets. However, Ebs1-loss in yeast or Smg7-depletion in human cells only partially disrupts NMD and in the latter, Smg7-depletion is partially compensated for by Smg6. Ebs1p physically interacts with the NMD helicase Upf1p and overexpressed Ebs1p leads to recruitment of Upf1p into cytoplasmic P-bodies. Furthermore, Ebs1p localizes to P-bodies upon glucose starvation along with Upf1p. Overall our findings suggest that NMD is more conserved in evolution than previously thought, and that at least one of the Smg5-7 proteins is conserved in budding yeast.

Nucleic Acids Research, 2013

Telomeres, the physical ends of eukaryotic chromosomes, are transcribed into telomeric repeatcont... more Telomeres, the physical ends of eukaryotic chromosomes, are transcribed into telomeric repeatcontaining RNA (TERRA), a large non-coding RNA, which forms an integral part of telomeric heterochromatin. In vitro, naked TERRA molecules are efficient inhibitors of human telomerase, base-pairing via their 5 0-UUAGGG-3 0 repeats with the template sequence of telomerase RNA, in addition to contacting the telomerase reverse transcriptase protein subunit. In vivo, however, TERRA-mediated inhibition of telomerase can be prevented by unknown mechanisms. Also, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) has been implicated in telomere length control. In vivo, TERRA is partially associated with hnRNPA1, and hnRNPA1 is also detected at telomeres. We demonstrate that on binding of TERRA, hnRNPA1 can alleviate the TERRA-mediated inhibition of telomerase. However, when in excess over TERRA, hnRNPA1 becomes itself an inhibitor of telomere extension, on binding of the telomeric DNA substrate. Yet, hnRNPA1 has no notable direct effects on the telomerase catalysis. Our in vitro results suggest that TERRA-mediated telomerase inhibition may be prevented by hnRNPA1 in vivo. Telomere extension by telomerase may require balanced levels of TERRA and hnRNPA1 at telomeres. Thus, TERRA and hnRNPA1 can function as a bimolecular regulator to turn telomerase and the telomere on and off.

Nucleic Acids Research, 2007

The human EST1A/SMG6 polypeptide physically interacts with the chromosome end replication enzyme ... more The human EST1A/SMG6 polypeptide physically interacts with the chromosome end replication enzyme telomerase. In an attempt to better understand hEST1A function, we have started to dissect the molecular interactions between hEST1A and telomerase. Here, we demonstrate that the interaction between hEST1A and telomerase is mediated by protein-RNA and protein-protein contacts. We identify a domain within hEST1A that binds the telomerase RNA moiety hTR while full-length hEST1A establishes in addition RNase-resistant and hTR-independent protein-protein contacts with the human telomerase reverse transcriptase polypeptide (TERT). Conversely, within hTERT, we identify a hEST1A interaction domain, which comprises hTR-binding activity and RNA-independent hEST1A-binding activity. Purified, recombinant hEST1A binds the telomerase RNA moiety (hTR) with high affinity (apparent overall K d = 25 nM) but low specificity. We propose that hEST1A assembles specifically with telomerase in the context of the hTR-hTERT ribonucleoprotein, through the high affinity of hEST1A for hTR and specific proteinprotein contacts with hTERT.

Molecular Cell, 2007

Telomerase is required for telomere maintenance and is responsible for the immortal phenotype of ... more Telomerase is required for telomere maintenance and is responsible for the immortal phenotype of cancer cells. How telomerase is assembled and reaches telomeres in the context of nuclear architecture is not understood. Recently, the telomerase RNA subunit (hTR) was shown to accumulate in Cajal bodies (CBs), subnuclear structures implicated in ribonucleoprotein maturation. However, the functional relevance of this localization for telomerase was unknown. hTR localization to CBs requires a short sequence motif called the CAB box. Here, we reconstitute telomerase in human cells and determine the effects of CAB box mutations on telomere biology. We demonstrate that mutant hTR, which fails to accumulate in CBs, is fully capable of forming catalytically active telomerase in vivo but is strongly impaired in telomere extension. The functional deficiency is accompanied by a decreased association of telomerase with telomeres. Collectively, these data identify subnuclear localization as an important regulatory mechanism for telomere length homeostasis in human cells.

Cell, 2003

Telomerase-mediated healing of broken chromosomes gives rise to terminal deletions and is repress... more Telomerase-mediated healing of broken chromosomes gives rise to terminal deletions and is repressed in most organisms. In ciliated protozoa, however, chromosome fragmentation and de novo telomere addition are part of the developmental program. Work by Karamysheva et al. (2003) in this issue of Cell indicates that in Euplotes crassus, this is mediated through switching between different telomerase reverse transcriptase isoforms.

Oncogene, 2001

Telomerase activation is crucial in human carcinogenesis. The limiting component of telomerase, t... more Telomerase activation is crucial in human carcinogenesis. The limiting component of telomerase, the catalytic subunit (hTERT), is undetectable in normal somatic cells but present in most tumor cells, including the earliest stages of colon carcinoma. The mechanisms involved in the dierential expression in normal and tumor cells are not understood. In normal cells hTERT expression is shut down by a repressor, and upregulation could be a consequence of cis-acting changes in the hTERT gene, making it resistant to repression. We have identi®ed a polymorphic and a monomorphic minisatellite in the second intron of the hTERT gene, and polymorphic one in intron 6. The polymorphic minisatellite in intron 2 contains binding sites for c-Myc, which has been shown to upregulate hTERT transcription. Screening colon carcinoma DNAs for rearrangements of hTERT minisatellites we detected no changes in 33 samples from tumors, most of which express hTERT. This indicates that size rearrangements of the hTERT minisatellites are not required for telomerase expression in colon carcinomas. Minor changes and one LOH were seen in ®ve tumors.

Nature genetics, 1999

The MYC proto-oncogene encodes a ubiquitous transcription factor (c-MYC) involved in the control ... more The MYC proto-oncogene encodes a ubiquitous transcription factor (c-MYC) involved in the control of cell proliferation and differentiation. Deregulated expression of c-MYC caused by gene amplification, retroviral insertion, or chromosomal translocation is associated with tumorigenesis. The function of c-MYC and its role in tumorigenesis are poorly understood because few c-MYC targets have been identified. Here we show that c-MYC has a direct role in induction of the activity of telomerase, the ribonucleoprotein complex expressed in proliferating and transformed cells, in which it preserves chromosome integrity by maintaining telomere length. c-MYC activates telomerase by inducing expression of its catalytic subunit, telomerase reverse transcriptase (TERT). Telomerase complex activity is dependent on TERT, a specialized type of reverse transcriptase. TERT and c-MYC are expressed in normal and transformed proliferating cells, downregulated in quiescent and terminally differentiated ce...

Ciba Foundation symposium, 1997

Telomerase, the enzyme that extends chromosomal DNA ends in most eukaryotes, contains essential R... more Telomerase, the enzyme that extends chromosomal DNA ends in most eukaryotes, contains essential RNA and protein subunits. We have been studying telomere replication in hypotrichous ciliates such as Euplotes aediculatus, which have numerous short macronuclear DNA molecules and therefore are highly enriched in telomeres and in telomerase. Cloning and sequencing genes for the RNA subunits from several ciliates revealed that telomerase RNAs with insignificant nucleotide sequence homology nevertheless form a common secondary structure. Affinity chromatography based on the sequence of the RNA subunit was used to purify the Euplotes telomerase as an active ribonucleoprotein enzyme. Two protein subunits, 123 kDa and 43 kDa, were identified. The finding of a yeast homologue to the 123 kDa subunit suggests that telomerase protein components may be much more highly conserved in evolution than the RNA subunits. The purified Euplotes telomerase has no activity with blunt-ended DNA primers, but i...

Biochemistry. Biokhimii͡a, 1997

Synthesis of telomeric repeats at chromosome ends requires telomerase, a ribonucleoprotein enzyme... more Synthesis of telomeric repeats at chromosome ends requires telomerase, a ribonucleoprotein enzyme. The RNA subunit, which contains the template for DNA synthesis, has been identified in many organisms. Recently, the protein subunit that catalyzes telomeric DNA extension has also been identified in Euplotes aediculatus and Saccharomyces cerevisiae. It has sequence and functional characteristics of a reverse transcriptase related to retrotransposon and retroviral reverse transcriptases, so this new family of telomerase subunits has been named TRT (Telomerase Reverse Transcriptase). We find it remarkable that the same type of protein structure required for retroviral replication is now seen to be essential for normal chromosome telomere replication in diverse eukaryotes.

Antisense oligonucleotides have been used to study the structure and function of small nuclear ri... more Antisense oligonucleotides have been used to study the structure and function of small nuclear ribonucleoprotein (snRNP) complexes and were adapted and modified for the purification of a variety of RNPs. We describe methods for recombinant expression and reconstitution of catalytically active human telomerase and the purification of native and recombinant telomerase using antisense affinity oligonucleotides. The purification procedure involves binding of the RNP complex to NeutrAvidin beads via a biotinylated 2'-O-methyl (2'-OMe) RNA oligonucleotide complementary to the RNA subunit. The complex is eluted from the beads through competition with a displacement oligonucleotide. Thus, the purified RNP is eluted under mild conditions, retaining its catalytic activity.

The EMBO Journal, 2000

Telomerase is the ribonucleoprotein enzyme responsible for the replication of chromosome ends in ... more Telomerase is the ribonucleoprotein enzyme responsible for the replication of chromosome ends in most eukaryotes. In the ciliate Euplotes aediculatus, the protein p43 biochemically co-puri®es with active telomerase and appears to be stoichiometric with both the RNA and the catalytic protein subunit of this telomerase complex. Here we describe cloning of the gene for p43 and present evidence that it is an authentic component of the telomerase holoenzyme. Comparison of the nucleotide sequence of the cloned gene with peptide sequences of the protein suggests that production of full-length p43 relies on a programmed ribosomal frameshift, an extremely rare translational mechanism. Anti-p43 antibodies immunodeplete telomerase RNA and telomerase activity from E.aediculatus nuclear extracts, indicating that the vast majority of mature telomerase complexes in the cell are associated with p43. The sequence of p43 reveals similarity to the La autoantigen, an RNAbinding protein involved in maturation of RNA polymerase III transcripts, and recombinant p43 binds telomerase RNA in vitro. By analogy to other La proteins, p43 may function in chaperoning the assembly and/or facilitating nuclear retention of telomerase.

Science, 2008

clicking here. colleagues, clients, or customers by , you can order high-quality copies for your ... more clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles

Science, 1995

... SCIENCE * VOL. 269 * 15 SEPTEMBER 1995 Telomerase and DNA End Replication: No Longer a Laggin... more ... SCIENCE * VOL. 269 * 15 SEPTEMBER 1995 Telomerase and DNA End Replication: No Longer a Lagging Strand Problem? Joachim Lingner, Julia Promisel Cooper, Thomas R. Cech 5' ....3' Tetomerase extends =overhang 3' 59 3rtSA

Proceedings of the National Academy of Sciences, 1996

Telomerase is a ribonucleoprotein enzyme that uses its internal RNA moiety as a template for synt... more Telomerase is a ribonucleoprotein enzyme that uses its internal RNA moiety as a template for synthesis of telomeric repeats at chromosome ends. Here we report the purification of'telomerase from Euplotes aediculatus by affinity chromatography with antisense 2'-O-methyl oligonucleotides, a method that was developed for small nuclear ribonucleoprotein particles (snRNPs). Elution of bound ribonucleoprotein from the antisense oligonucleotide under nondenaturing conditions was achieved by a novel approach, using a displacement oligonucleotide. Polypeptides of 12Q kDa and 43 kDa (a doublet) copurify with the active telomerase and appear stoichiometric with telomerase RNA. A simple model for DNA end replication predicts that after semiconservative DNA replication, telomerase will extend the newly synthesized, blunt-ended leading strand. We show that purified Euplotes telomerase has no activity with blunt-ended primers. Instead, efficient extension requires 4 to 6 single-stranded nucleotides at the 3' end. Therefore, this model predicts the existence of other activities such as helicases or nucleases that generate a single-stranded 3' end from a blunt end, thus'activating the end for telomerase extension.

Nucleic Acids Research, 1993

Two commonly used methods to end-label RNAmolecules are 5'-end labeling by polynucleotlde klnase ... more Two commonly used methods to end-label RNAmolecules are 5'-end labeling by polynucleotlde klnase and 3'-end labeling with pCp and T4 RNA llgase. We show here that RNA 3'-ends can also be labeled with the chain-terminating analogue cordycepln 5'-trlphosphate (3'-deoxy-ATP) which Is added by poly(A) polymerase. For a synthetic RNA It Is shown that 40% of cordycepin becomes Incorporated when the nucleotide Is used at limiting concentrations and that with an excess of cordycepin 5'-triphosphate essentially all the RNA becomes modified at Its 3'-end. The reaction Is complete within minutes and the RNA product is uniform and suitable for sequence analysis. The efficiency of labeling varies with different RNAmolecules and is different from RNA ligase. Poly(A) polymerase preferentially labels longer RNA-molecules whereas short RNA-molecules are labeled more efficiently by T4 RNA llgase.

Nucleic Acids Research, 2010

Telomeres, the physical ends of eukaryotes chromosomes are transcribed into telomeric repeat cont... more Telomeres, the physical ends of eukaryotes chromosomes are transcribed into telomeric repeat containing RNA (TERRA), a large non-coding RNA of unknown function, which forms an integral part of telomeric heterochromatin. TERRA molecules resemble in sequence the telomeric DNA substrate as they contain 5 0-UUAGGG-3 0 repeats near their 3 0-end which are complementary to the template sequence of telomerase RNA. Here we demonstrate that endogenous TERRA is bound to human telomerase in cell extracts. Using in vitro reconstituted telomerase and synthetic TERRA molecules we demonstrate that the 5 0-UUAGGG-3 0 repeats of TERRA base pair with the RNA template of the telomerase RNA moiety (TR). In addition TERRA contacts the telomerase reverse transcriptase (TERT) protein subunit independently of hTR. In vitro studies further demonstrate that TERRA is not used as a telomerase substrate. Instead, TERRA acts as a potent competitive inhibitor for telomeric DNA in addition to exerting an uncompetitive mode of inhibition. Our data identify TERRA as a telomerase ligand and natural direct inhibitor of human telomerase. Telomerase regulation by the telomere substrate may be mediated via its transcription.

Nucleic Acids Research, 2007

The Smg proteins Smg5, Smg6 and Smg7 are involved in nonsense-mediated RNA decay (NMD) in metazoa... more The Smg proteins Smg5, Smg6 and Smg7 are involved in nonsense-mediated RNA decay (NMD) in metazoans, but no orthologs have been found in the budding yeast Saccharomyces cerevisiae. Sequence alignments reveal that yeast Ebs1p is similar in structure to the human Smg5-7, with highest homology to Smg7. We demonstrate here that Ebs1p is involved in NMD and behaves similarly to human Smg proteins. Indeed, both loss and overexpression of Ebs1p results in stabilization of NMD targets. However, Ebs1-loss in yeast or Smg7-depletion in human cells only partially disrupts NMD and in the latter, Smg7-depletion is partially compensated for by Smg6. Ebs1p physically interacts with the NMD helicase Upf1p and overexpressed Ebs1p leads to recruitment of Upf1p into cytoplasmic P-bodies. Furthermore, Ebs1p localizes to P-bodies upon glucose starvation along with Upf1p. Overall our findings suggest that NMD is more conserved in evolution than previously thought, and that at least one of the Smg5-7 proteins is conserved in budding yeast.

Nucleic Acids Research, 2013

Telomeres, the physical ends of eukaryotic chromosomes, are transcribed into telomeric repeatcont... more Telomeres, the physical ends of eukaryotic chromosomes, are transcribed into telomeric repeatcontaining RNA (TERRA), a large non-coding RNA, which forms an integral part of telomeric heterochromatin. In vitro, naked TERRA molecules are efficient inhibitors of human telomerase, base-pairing via their 5 0-UUAGGG-3 0 repeats with the template sequence of telomerase RNA, in addition to contacting the telomerase reverse transcriptase protein subunit. In vivo, however, TERRA-mediated inhibition of telomerase can be prevented by unknown mechanisms. Also, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) has been implicated in telomere length control. In vivo, TERRA is partially associated with hnRNPA1, and hnRNPA1 is also detected at telomeres. We demonstrate that on binding of TERRA, hnRNPA1 can alleviate the TERRA-mediated inhibition of telomerase. However, when in excess over TERRA, hnRNPA1 becomes itself an inhibitor of telomere extension, on binding of the telomeric DNA substrate. Yet, hnRNPA1 has no notable direct effects on the telomerase catalysis. Our in vitro results suggest that TERRA-mediated telomerase inhibition may be prevented by hnRNPA1 in vivo. Telomere extension by telomerase may require balanced levels of TERRA and hnRNPA1 at telomeres. Thus, TERRA and hnRNPA1 can function as a bimolecular regulator to turn telomerase and the telomere on and off.

Nucleic Acids Research, 2007

The human EST1A/SMG6 polypeptide physically interacts with the chromosome end replication enzyme ... more The human EST1A/SMG6 polypeptide physically interacts with the chromosome end replication enzyme telomerase. In an attempt to better understand hEST1A function, we have started to dissect the molecular interactions between hEST1A and telomerase. Here, we demonstrate that the interaction between hEST1A and telomerase is mediated by protein-RNA and protein-protein contacts. We identify a domain within hEST1A that binds the telomerase RNA moiety hTR while full-length hEST1A establishes in addition RNase-resistant and hTR-independent protein-protein contacts with the human telomerase reverse transcriptase polypeptide (TERT). Conversely, within hTERT, we identify a hEST1A interaction domain, which comprises hTR-binding activity and RNA-independent hEST1A-binding activity. Purified, recombinant hEST1A binds the telomerase RNA moiety (hTR) with high affinity (apparent overall K d = 25 nM) but low specificity. We propose that hEST1A assembles specifically with telomerase in the context of the hTR-hTERT ribonucleoprotein, through the high affinity of hEST1A for hTR and specific proteinprotein contacts with hTERT.

Molecular Cell, 2007

Telomerase is required for telomere maintenance and is responsible for the immortal phenotype of ... more Telomerase is required for telomere maintenance and is responsible for the immortal phenotype of cancer cells. How telomerase is assembled and reaches telomeres in the context of nuclear architecture is not understood. Recently, the telomerase RNA subunit (hTR) was shown to accumulate in Cajal bodies (CBs), subnuclear structures implicated in ribonucleoprotein maturation. However, the functional relevance of this localization for telomerase was unknown. hTR localization to CBs requires a short sequence motif called the CAB box. Here, we reconstitute telomerase in human cells and determine the effects of CAB box mutations on telomere biology. We demonstrate that mutant hTR, which fails to accumulate in CBs, is fully capable of forming catalytically active telomerase in vivo but is strongly impaired in telomere extension. The functional deficiency is accompanied by a decreased association of telomerase with telomeres. Collectively, these data identify subnuclear localization as an important regulatory mechanism for telomere length homeostasis in human cells.

Cell, 2003

Telomerase-mediated healing of broken chromosomes gives rise to terminal deletions and is repress... more Telomerase-mediated healing of broken chromosomes gives rise to terminal deletions and is repressed in most organisms. In ciliated protozoa, however, chromosome fragmentation and de novo telomere addition are part of the developmental program. Work by Karamysheva et al. (2003) in this issue of Cell indicates that in Euplotes crassus, this is mediated through switching between different telomerase reverse transcriptase isoforms.