Biology subjects

Zaug, A. J.

Publications and source records attributed to Zaug, A. J..

7 recordsLinked to original sources

bioRxiv2026

Thiomorpholino antisense oligonucleotides inhibit telomerase and limit cancer cell proliferation

Reactivation of telomerase confers immortality to approximately 90% of human tumors by enabling continuous elongation of the DNA at chromosome ends, or telomeres. The telomerase catalytic subunit TERT adds TTAGGG repeats using a portion of the telomerase RNA component hTR as a template. Because telomerase is inactive in most normal somatic cells, it remains an attractive therapeutic target; however, no telomerase inhibitor has yet demonstrated robust clinical efficacy with acceptable safety. Here we evaluate thiomorpholino oligonucleotides (TMOs) as a new class of antisense oligonucleotides targeting the template region of hTR. TMOs incorporate morpholino rings and phosphorothioate linkages, which enhance nuclease resistance, RNA binding and nuclear uptake. Two anti-hTR TMOs inhibited telomerase activity in vitro with an IC50 below 1 nM, whereas two control TMOs were at least 100-fold less active. HeLa cells treated with anti-hTR TMOs showed progressive telomere shortening, detectable after one week of treatment. Growth inhibition was observed after substantial telomere erosion, and both telomere length and proliferation recovered upon withdrawal of TMOs. These findings establish TMOs as a promising new chemistry for telomerase-targeted therapeutics.

molecular biology↗

bioRxiv2026

Impaired synthesis of both telomere strands and adaptive TERT reduction in RTEL1 ATPase-dead cells

Regulator of Telomere Elongation Helicase 1 (RTEL1) plays a critical role in telomere replication by disassembling DNA secondary structures such as G-quadruplexes and telomeric loops (T-loops). However, its precise mechanism remains unclear. Previously, we generated HeLa cells homozygous for ATPase-dead RTEL1. Here, we found that these cells have a 50-60% reduction in the synthesis of both telomere strands, indicating that RTEL1 ATPase activity is essential for both leading-strand and lagging-strand synthesis. Surprisingly, these cells also showed a substantial reduction in Telomerase Reverse Transcriptase (TERT) mRNA levels and a 60-80% reduction in telomerase activity, without activating alternative lengthening of telomeres (ALT). Forced TERT overexpression suppressed proliferation and caused late S/G2 accumulation, implying that the natural TERT reduction provides adaptive resistance. Expressing wild-type (WT) RTEL1 at levels close to physiological levels failed to rescue growth defects, suggesting a dominant-negative effect. These results reveal unexpected interactions between RTEL1 and telomerase and show how cancer cells compensate for RTEL1 dysfunction by decreasing TERT expression.

cell biology↗

bioRxiv2026

Human vault RNAs exhibit diverse expression patterns and inter-locus compensation

In 1986, a class of small, noncoding RNAs was discovered in association with an enormous, enigmatic ribonucleoprotein complex - the "vault" particle - and thus dubbed vault RNAs (vtRNAs). However, its since been recognized that the vast majority ([≥]95%) of these noncoding vtRNA molecules are not associated with the mysterious vaults, raising questions about their potential independent function(s). Moreover, humans express four vtRNAs from two different loci, and debate has arisen about whether the vtRNA paralogs share any functional connection, and whether they should remain classified together. Herein, we report the expression patterns of the four VTRNA paralogs in a variety of human cell lines, including various single- and multi-gene VTRNA-knockout cell lines. Knockout of one or more of the three VTRNA1 genes leads to increased expression of vtRNA2-1, suggesting that their biological functions are related. Additionally, we interrogated the effects of vtRNA knockout on HEK293T cell growth, viability, and viral infection, and were unable to replicate previously reported associations. Collectively, our findings point to a potential functional connection between even the most distantly related human vtRNA paralogs, while reaffirming that their biological roles and mechanisms still require critical study.

molecular biology↗

bioRxiv2023

Dissecting telomerase RNA structural heterogeneity in living human cells with DMS-MaPseq

Telomerase is a specialized reverse transcriptase that uses an intrinsic RNA subunit as the template for telomeric DNA synthesis. Biogenesis of human telomerase requires its RNA subunit (hTR) to fold into a multi-domain architecture that includes the template-containing pseudoknot (t/PK) and the three-way junction (CR4/5). These two hTR domains bind the telomerase reverse transcriptase (hTERT) protein and are thus essential for telomerase catalytic activity. Here, we probe the structure of hTR in living cells using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and ensemble deconvolution analysis. Unexpectedly, approximately 15% of the steady state population of hTR has a CR4/5 conformation lacking features thought to be required for hTERT binding. The proportion of hTR CR4/5 that is folded into the primary functional conformation does not require hTERT expression and the fraction of hTR that assumes a misfolded CR4/5 domain is not refolded by overexpression of its hTERT binding partner. This result suggests a functional role for an RNA folding cofactor other than hTERT during telomerase biogenesis. Mutagenesis demonstrates that stabilization of the alternative CR4/5 conformation is detrimental to telomerase assembly and activity. Moreover, the alternative CR4/5 conformation is not found in telomerase RNP complexes purified from cells via an epitope tag on hTERT, supporting the hypothesis that only the major CR4/5 conformer is active. We propose that this misfolded portion of the cellular hTR pool is either slowly refolded or degraded. Thus, kinetic traps for RNA folding that have been so well-studied in vitro may also present barriers for assembly of ribonucleoprotein complexes in vivo.

biochemistry↗

bioRxiv2021

CST does not evict elongating telomerase but prevents initiation by ssDNA binding

The CST complex (CTC1-STN1-TEN1) has been shown to inhibit telomerase extension of the G-strand of telomeres and facilitate the switch to C-strand synthesis by DNA polymerase alpha-primase (pol -primase). Recently the structure of human CST was solved by cryo-EM, allowing the design of mutant proteins defective in telomeric ssDNA binding and prompting the reexamination of CST inhibition of telomerase. The previous proposal that human CST inhibits telomerase by sequestration of the DNA primer was tested with a series of DNA-binding mutants of CST and modeled by a competitive binding simulation. The DNA-binding mutants had substantially reduced ability to inhibit telomerase, as predicted from their reduced affinity for telomeric DNA. These results provide strong support for the previous primer sequestration model. We then tested whether addition of CST to an ongoing processive telomerase reaction would terminate DNA extension. Pulse-chase telomerase reactions with addition of either wild-type CST or DNA-binding mutants showed that CST has no detectable ability to terminate ongoing telomerase extension in vitro. The same lack of inhibition was observed with or without pol -primase bound to CST. These results suggest how the switch from telomerase extension to C-strand synthesis may occur.

biochemistry↗

bioRxiv2019

The cryo-EM structure of human CST reveals a two-megadalton decameric assembly bound to telomeric DNA

The single-stranded DNA-binding CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and genome-wide replication recovery, processes that are critical for genome stability. Here, we report the 2.95 [A] cryo-EM structure of human CST bound to telomeric single-stranded DNA, which unexpectedly assembles as a decameric supercomplex. The atomic model of the 134 kDa CTC1, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. In situ arrangements of STN1 and TEN1 are revealed, with STN1 interacting with CTC1 at two separated sites, allowing allosteric mediation of CST decameric assembly. Surprisingly, CTC1 lacks the anticipated structural homology to yeast Cdc13 but instead shares similarity with a form of Replication Protein A. The atomic-resolution model of human CST provides crucial mechanistic understanding of CST mutations associated with human diseases. Moreover, the decameric form of CST suggests the intriguing possibility of ssDNA architectural organization similar to what the nucleosome provides for dsDNA.\n\nOne Sentence SummaryHuman telomeric single-stranded DNA triggers the assembly of a decameric protein supercomplex solved by cryo-EM.

biophysics↗

bioRxiv2017

Dynamics of human telomerase recruitment depend on template-telomere base-pairing

The reverse transcriptase telomerase adds telomeric repeats to chromosome ends to counteract telomere shortening and thereby assures genomic stability in dividing human cells. Key variables in telomere homeostasis are the frequency with which telomerase engages the chromosome end and the number of telomeric repeats it adds during each association event. To study telomere elongation in vivo we have established a live-cell imaging assay to track individual telomerase RNPs in HeLa cells. Using this assay and the drug imetelstat, which is a competitive inhibitor of telomeric DNA binding, we demonstrate that stable association of telomerase with the single-stranded overhang of the chromosome end requires telomerase-DNA base-pairing. Furthermore, we show that telomerase processivity contributes to telomere elongation in vivo. Together, these findings provide new insight into the dynamics of telomerase recruitment and the importance of processivity in maintaining telomere length in human cancer cells.

cell biology↗