Telomeres which are present at the ends of eukaryotic chromosomes mediate genome stability and determine cellular lifespan 1 . Telomeric repeat containing RNAs (TERRA) are long noncoding RNAs transcribed from chromosome ends 2 , 3 , which regulate telomeric chromatin structure and telomere maintenance via telomerase and homology directed repair (HDR) 4 , 5 . The mechanisms by which TERRA is recruited to chromosome ends remain poorly defined. Here we develop a reporter system to dissect the underlying mechanisms and demonstrate 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 triggers telomere fragility and is promoted by the RAD51 recombinase and its interacting partner BRCA2 but counteracted by RNA surveillance factors, RNaseH1 and TRF1. RAD51 physically interacts with TERRA and catalyzes R-loop formation with TERRA in vitro supporting a direct involvement of this DNA recombinase in TERRA recruitment by strand invasion. Together, our findings reveal a RAD51-dependent pathway that governs TERRA mediated R-loop formation post transcription providing a mechanism of how lncRNAs can be recruited to new loci in trans .

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.

Material and Methods Analysis of methylated DNA Cells were seeded at a density of 40.000 cell/ml and transfected with siCEBPB-AS or siControl as described in the M&M. DNA was extracted after 48 h transfection using the QIAamp DNA Mini Kit (Qiagen). NEBuffer (New England BioLabs), supplemented with GTP and BSA, was added to 200 ng of each DNA sample. Samples were either treated with MrcBC enzyme (New England BioLabs) or mock treated, and incubated overnight at 37C. Next day, the enzyme was heat inactivated at 65C° for 1 hour. The enzyme MrcBC is a methylation-dependent endonuclease that cleaves methylated DNA at Pu m CG sequence elements. Samples were subjected to RT-qPCR and standardized to uncut input. Delta CT values were converted to fold-change values and the ratio between siCEBPB-AS/siControl were calculated.