RAD51-dependent recruitment of TERRA lncRNA to telomeres through R-loops

Telomeres-repeated, noncoding nucleotide motifs and associated proteins that are found at the ends of eukaryotic chromosomes-mediate genome stability and determine cellular lifespan1. Telomeric-repeat-containing RNA (TERRA) is a class of long noncoding RNAs (lncRNAs) that are transcribed from chromosome ends2,3; these RNAs in turn regulate telomeric chromatin structure and telomere maintenance through the telomere-extending enzyme telomerase4-6 and homology-directed DNA repair7,8. The mechanisms by which TERRA is recruited to chromosome ends remain poorly defined. Here we develop a reporter system with which to dissect the underlying mechanisms, and show that the UUAGGG repeats of TERRA are both necessary and sufficient to target TERRA to chromosome ends. TERRA preferentially associates with short telomeres through the formation of telomeric DNA-RNA hybrid (R-loop) structures that can form in trans. Telomere association and R-loop formation trigger telomere fragility and are promoted by the recombinase RAD51 and its interacting partner BRCA2, but counteracted by the RNA-surveillance factors RNaseH1 and TRF1. RAD51 physically interacts with TERRA and catalyses R-loop formation with TERRA in vitro, suggesting a direct involvement of this DNA recombinase in the recruitment of TERRA by strand invasion. Together, our findings reveal a RAD51-dependent pathway that governs TERRA-mediated R-loop formation after transcription, providing a mechanism for the recruitment of lncRNAs to new loci in trans.

RADX: A novel single-stranded DNA-binding protein regulating telomere recombination

Anna-Sophia Briod

Telomeres are specialized nucleoprotein structures present at the ends of linear chromosome. The telomeric DNA part is comprised of 5-15 kilo base pairs of double stranded TTAGGG repeats and it contains at the 3' end a single-stranded G-rich overhang of 50-400 nucleotides. Telomeric DNA associates with a large number of proteins, which inhibit DNA damage checkpoint activation, DNA repair activities and nucleolytic degradation. To better understand the complex roles which telomeres play in genome stability, premature aging and cancer development we explored the telomeric proteomic environment with BioID. We employed a CRISPR/Cas9 based knock-in approach to integrate the promiscuous biotin ligase BirA at the genomic loci of the telomeric components TRF1, TRF2 and POT1. Thus, we expressed the fusion proteins from native promoters aiming at retaining native expression levels. Upon incubation with Biotin, the promiscuous biotinylase can label proximal proteins, which can be selectively purified and identified via mass spectrometry. Our BioID results indicated that TRF1, TRF2 and POT1 share a large number of common protein partners. In addition to already known telomere components, we identified a significant number of novel telomeric proteins. Among those newly identified proteins, we found RADX, an RPA-like single-stranded DNA-binding-protein, which counteracts RAD51 at stalled replication forks. Even though RADX had so far only been characterized for its role in DNA replication we observed colocalization of ectopically expressed 3xFlag-RADX with telomeres throughout the cell cycle. The colocalization decreased when the RADXs single-stranded (ss)DNA-binding domain was mutated. We therefore hypothesize that RADX binds the single-stranded G-rich telomeric strand either as displaced strand in the t-loop or as single-stranded telomeric overhang. Chromatin-immunoprecipitation experiments suggest that RADX plays a distinct role in telomere replication, since upon treatment with the replication stress inducing reagent hydroxyurea we observed increased binding of RADX specifically to telomeres. Notably, RADX depletion on its own did not alter telomere integrity and telomere length but upon co-depletion with POT1, telomere fragility, sister-chromatid associations and telomere length increased strikingly. POT1 and POT1/RADX depletions lead to increased associations of the recombinase RAD51 with telomeres and consequently silencing of RAD51 rescued telomere integrity and elongation implying the regulatory role of RADX for RAD51. In summary my thesis describes the proteomic microenvironment of different telomeric proteins, identifies the novel telomeric protein RADX and characterizes its role at telomeres. My findings provide important insights into how homologous recombination and RAD51 loading is suppressed at telomeres.

EPFL2019

A three-state model for the regulation of telomerase by TERRA and hnRNPA1

Joachim Lingner, Sophie Redon, Ivo Zemp

Telomeres, the physical ends of eukaryotic chromosomes, are transcribed into telomeric repeat-containing 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'-UUAGGG-3' 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.

Oxford University Press2013

The makings of TERRA R-loops at chromosome ends

Marianna Feretzaki, Joachim Lingner, Rita Valador Fernandes

Telomeres protect chromosome ends from nucleolytic degradation, uncontrolled recombination by DNA repair enzymes and checkpoint signaling, and they provide mechanisms for their maintenance by semiconservative DNA replication, telomerase and homologous recombination. The telomeric long noncoding RNA TERRA is transcribed from a large number of chromosome ends. TERRA has been implicated in modulating telomeric chromatin structure and checkpoint signaling, and in telomere maintenance by homology directed repair, and telomerase - when telomeres are damaged or very short. Recent work indicates that TERRA association with telomeres involves the formation of DNA:RNA hybrid structures that can be formed post transcription by the RAD51 DNA recombinase, which in turn may trigger homologous recombination between telomeric repeats and telomere elongation. In this review, we describe the mechanisms of TERRA recruitment to telomeres, R-loop formation and its regulation by shelterin proteins. We discuss the consequences of R-loop formation, with regard to telomere maintenance by DNA recombination and how this may impinge on telomere replication while counteracting telomere shortening in normal cells and in ALT cancer cells, which maintain telomeres in the absence of telomerase.

TAYLOR & FRANCIS INC2021

RADX: A novel single-stranded DNA-binding protein regulating telomere recombination

Anna-Sophia Briod

EPFL2019