UUAGGG Repeats Research Articles

DNA-RNA hybrid G-quadruplex tends to form near the 3′ end of telomere overhang

Telomeric repeat-containing RNA (TERRA) has been suggested to participate in telomere maintenance. TERRA consisting of UUAGGG repeats is capable of forming an intermolecular G-quadruplex (GQ) with single-stranded TTAGGG-repeat DNA in the telomere 3′ overhang. To explore the structural features and potential functions of this DNA-RNA hybrid GQ (HGQ), we used single-molecule FRET to study the folding patterns of DNA with four to seven telomeric tandem repeats annealed with a short RNA consisting of two or five telomeric repeats. Our data highlight that RNA prefers to form DNA-RNA HGQ near the 3′ end of telomeric DNA. Furthermore, the unfolding of secondary structures by a complementary C-rich sequence was observed for DNA GQ but not for DNA-RNA HGQ, which demonstrated the enhanced stability of the telomere 3′ end via hybridization with RNA. These conformational and physical properties of telomeric DNA-RNA HGQ suggest that TERRA might limit access to the 3′ end of the telomeric DNA overhang, which is known to be critical for the interaction with telomerase and other telomere-associated proteins.

Abstract 2743: Telomere transcripts improve synthetic inhibitors of telomerase

Abstract Most tumors express telomerase for infinite cell growth capacity. Telomerase was recognized recently as central regulator of all of the hallmarks of cancer and synthetic inhibitors of telomerase, such as Imetelstat blocking the RNA subunit of telomerase TERC, are in clinical trials. RNA transcripts from telomeres called TERRA were identified to block telomerase activity (TA) potentially via direct binding to TERC. We established recombinant expression systems to modulate TERRA transcripts in cell culture cells and study in vitro cell growth and viability directly and in combination with telomerase inhibitors. Adeno- and lentivirus constructs (AV and LV) were established for transient and stable recombinant TERRA expression in TA tumor cell lines (N=3) and mortal fibroblast cells as control. AV and LV express TERRA under control of hH1 promoter with around 120 units of UUAGGG repeats in sense (S) or anti-sense (AS) orientation. Similarly, AV and LV express polyadenylated form of TERRA under control of CMV promoter and polyadenylation signaling sites. Telomere length (TL), endogenous and recombinant TERRA expression was analyzed by qPCR, TA by TRAP assay. Population doubling (PD) times were calculated from cell growth numbers of LV clones. Small molecule telomerase inhibitors and AVs expressing dominant-negative telomerase and shRNA against hTERT were used in MTT cell viability and clonogenicity assays. TERRA expression was modulated transiently and stably by recombinant AVs and LVs as compared to virus controls expressing eGFP. Moderate 1.5-1.7 fold elevation of recombinant TERRA-S transcripts caused reduction of TA to 23-38%. In contrast, AS expression reduced TERRA 3-8 fold without reduction of TA. TL, PD time, cell viability and clonogenicity were not affected by TERRA-S and -AS expression up to 3 weeks in culture. Single cell clones were isolated from cells infected by LVs. Significant improve of telomerase targeting approaches was identified as IC-50 values decreased 2.0-2.6 fold in LV cell clones expressing moderate recombinant TERRA-S compared to TERRA-AS and eGFP. Similarly, clone formation capacity decreased 1.3-1.7 fold. Human fibroblasts showed 15 fold increased TERRA expression compared to tumor cells and were not affected by treatments applied. Preliminary data with modified RNA oligonucleotides containing TERRA sequences indicate that up-take by cells was efficient. Furthermore, IC-50 values of TA inhibitor MST-312 in combination with 3 nM TERRA oligonucleotides decreased 1.9-3.9 fold compared to mismatch controls. Effective concentrations were 1000 fold lower compared to DNA oligonucleotide telomerase inhibitors currently in clinical trials. We demonstrated that recombinant expression of TERRA significantly improved telomerase targeting approaches in tumor cells but not in mortal fibroblasts. TERRA oligonucleotides are candidates for in vivo application in combination with telomerase targeting approaches or other tumor therapies. Citation Format: Sandra Sampl, Doris Mejri, Christian Stern, Hui Wang, Klaus Holzmann. Telomere transcripts improve synthetic inhibitors of telomerase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2743. doi:10.1158/1538-7445.AM2014-2743

Structure of Human Telomeric RNA (TERRA): Stacking of Two G-Quadruplex Blocks in K+ Solution

Telomeric repeat-containing RNAs (TERRA) are transcription products of the telomeres. Human TERRA sequences containing UUAGGG repeats can form parallel-stranded G-quadruplexes. The stacking interaction of such structures was shown to be important for ligand targeting and higher-order arrangement of G-quadruplexes in long TERRA sequences. Here we report on the first high-resolution structure of a stacked G-quadruplex formed by the 10-nucleotide human TERRA sequence r(GGGUUAGGGU) in potassium solution. This structure comprises two dimeric three-layer parallel-stranded G-quadruplex blocks, which stack on each other at their 5'-ends. The adenine in each UUA loop is nearly coplanar with the 5'-end G-tetrad forming an A·(G·G·G·G)·A hexad, thereby increasing the stacking contacts between the two blocks. Interestingly, this stacking and loop conformation is different from all structures previously reported for the free human TERRA but resembles the structure previously determined for a complex between a human TERRA sequence and an acridine ligand. This stacking conformation is a potential target for drugs that recognize or induce the stacking interface.

More on the Lack of Correlation between Terra Expression and Telomere Length

More on the Lack of Correlation between Terra Expression and Telomere Length

Oligonucleotide Models of Telomeric DNA and RNA Form a Hybrid G-quadruplex Structure as a Potential Component of Telomeres

Telomeric repeat-containing RNA, a non-coding RNA molecule, has recently been found in mammalian cells. The detailed structural features and functions of the telomeric RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In this study, using model human telomeric DNA and RNA sequences, we demonstrated that human telomeric RNA and DNA oligonucleotides form a DNA-RNA G-quadruplex. We next employed chemistry-based oligonucleotide probes to mimic the naturally formed telomeric DNA-RNA G-quadruplexes in living cells, suggesting that the process of DNA-RNA G-quadruplex formation with oligonucleotide models of telomeric DNA and RNA could occur in cells. Furthermore, we investigated the possible roles of this DNA-RNA G-quadruplex. The formation of the DNA-RNA G-quadruplex causes a significant increase in the clonogenic capacity of cells and has an effect on inhibition of cellular senescence. Here, we have used a model system to provide evidence about the formation of G-quadruplex structures involving telomeric DNA and RNA sequences that have the potential to provide a protective capping structure for telomere ends.

Structure of Long Human Telomeric RNA (TERRA): G-Quadruplexes Formed by Four and Eight UUAGGG Repeats Are Stable Building Blocks

The discovery of long RNA transcripts of telomeric repeats (TERRA) and their potential to form G-quadruplexes stimulated studies on the possible arrangements of G-quadruplexes along TERRA. Here we performed ribonuclease protection assay to investigate the structures formed by long human TERRA. We found that G-quadruplexes comprising four and eight UUAGGG repeats were most resistant to RNase T1 digestion, presumably with the former adopting an all-parallel-stranded propeller-type conformation and the latter forming a structure with two tandemly stacked G-quadruplex subunits each containing three G-tetrad layers. Molecular dynamics simulations of eight-repeat human TERRA sequences consisting of different stacking interfaces between the two G-quadruplex subunits, i.e., 5'-5', 3'-3', 3'-5', and 5'-3', demonstrated stacking feasibility for all but the 5'-3' arrangement. A continuous stacking of the loop bases from one G-quadruplex subunit to the next was observed for the 5'-5' stacking conformation. We also put forward other possible stacking arrangements that involve more than one linker connecting the two G-quadruplex subunits. On the basis of these results, we propose a "beads-on-a-string"-like arrangement along human TERRA, whereby each bead is made up of either four or eight UUAGGG repeats in a one- or two-block G-quadruplex arrangement, respectively.

Molecular Dissection of Telomeric Repeat-Containing RNA Biogenesis Unveils the Presence of Distinct and Multiple Regulatory Pathways

Telomeres are transcribed into telomeric repeat-containing RNA (TERRA), large, heterogeneous, noncoding transcripts which form part of the telomeric heterochromatin. Despite a large number of functions that have been ascribed to TERRA, little is known about its biogenesis. Here, we present the first comprehensive analysis of the molecular structure of TERRA. We identify biochemically distinct TERRA complexes, and we describe TERRA regulation during the cell cycle. Moreover, we demonstrate that TERRA 5' ends contain 7-methylguanosine cap structures and that the poly(A) tail, present on a fraction of TERRA transcripts, contributes to their stability. Poly(A)(-) TERRA, but not poly(A)(+) TERRA, is associated with chromatin, possibly reflecting distinct biological roles of TERRA ribonucleoprotein complexes. In support of this idea, poly(A)(-) and poly(A)(+) TERRA molecules end with distinct sequence registers. We also determine that the bulk of 3'-terminal UUAGGG repeats have an average length of 200 bases, indicating that the length heterogeneity of TERRA likely stems from its subtelomeric regions. Finally, we find that TERRA is regulated during the cell cycle, being lowest in late S phase and peaking in early G(1). Our analyses offer the basis for investigating multiple regulatory pathways that affect TERRA synthesis, processing, turnover, and function.

Developmentally regulated transcription of mammalian telomeres by DNA-dependent RNA polymerase II

Mammalian telomeres consist of non-coding TTAGGG repeats that are bound by the multi-protein complex 'shelterin', thus protecting chromosome ends from DNA repair mechanisms and degradation. Mammalian telomeric chromatin is enriched for the constitutive heterochromatin marks H3K9me3, H4K20me3 and HP1 (refs 2, 3, 4, 5, 6, 7). Similar to pericentric heterochromatin, telomeric heterochromatin is thought to be fundamental for the maintenance of chromosomal integrity. Here, we report that telomeric repeats are transcribed by DNA-dependent RNA polymerase II, which, in turn, interacts with the TRF1 shelterin protein. Telomeric RNAs (TelRNAs) contain UUAGGG repeats, are polyadenylated and are transcribed from the telomeric C-rich strand. Transcription of mammalian telomeres is regulated by several mechanisms, including developmental status, telomere length, cellular stress, tumour stage and chromatin structure. Using RNA-flourescent in situ hybridization (FISH), we show that TelRNAs are novel structural components of telomeric chromatin. Importantly, we provide evidence that TelRNAs block the activity of telomerase in vitro, suggesting that TelRNAs may regulate telomerase activity at chromosome ends. Our results indicate that TelRNAs are novel components of mammalian telomeres, which are anticipated to be fundamental for understanding telomere biology and telomere-related diseases, such as cancer and ageing.

A Xenopus egg factor with DNA-binding properties characteristic of terminus-specific telomeric proteins.

We have identified a Xenopus laevis protein factor that specifically recognizes vertebrate telomeric repeats at DNA ends. This factor, called Xenopus telomere end factor (XTEF), is detected predominantly in extracts of Xenopus eggs and ovaries, which are estimated to contain sufficient XTEF to bind approximately 3 x 10(7) DNA ends. In contrast, XTEF is much less abundant (approximately 90 per cell) in extracts of somatic cell nuclei. Mobility retardation analysis of the XTEF activity in egg extracts indicates that this factor binds the vertebrate telomeric repeat sequence (TTAGGG)2 when present in a single-stranded 3' overhang. Single-stranded 3' extensions of (TTTGGG)2, (AAAGGG)2, (TTACCC)2, or a nonrepetitive sequence fail to bind XTEF efficiently, whereas changes in the double-stranded sequence 5' to the TTAGGG repeat tail are tolerated. TTAGGG repeats are not recognized at internal position, at a 5' protruding end, or in double-stranded DNA. In addition, the factor does not bind RNA with single-stranded UUAGGG repeats at a 3' end. XTEF-DNA complexes form and are stable in high salt. The DNA-binding properties of XTEF resemble the characteristics of a class of terminus-specific telomere proteins identified previously in hypotrichous ciliates.