Regulation and impact of TERRA R-loops at human telomeres

Telomeres are the nucleoprotein structures found at the ends of linear chromosomes. They ensure that the termini of chromosomes are not inappropriately recognized as sites of DNA damage, and are therefore crucial for genome stability. In spite of the heterochromatin-characteristic features present at telomeres and subtelomeric regions, telomeres are transcribed into the telomeric repeat containing RNA - TERRA.The transcription of TERRA stems from promoters residing within subtelomeric sequences in most chromosome arms. It proceeds through the telomeric repetitive tract, using the C-rich telomeric strand as template. TERRA has been implicated in multiple processes, such as the regulation of the heterochromatic structure of telomeres, and the modulation of telomerase - the ribonucleoprotein complex which extends telomeric DNA. Notably, TERRA is capable of hybridizing telomeric DNA, forming three-stranded structures termed R-loops, which comprise a DNA:RNA hybrid and a displaced DNA strand. Remarkably, many functions attributed to TERRA have been shown to depend on TERRA R-loops, including the stimulation of telomere elongation mediated by homologous-recombination, as well as the interference with the progression of the DNA replication machinery for appropriate duplication of telomeric DNA.Aiming to elucidate the regulation of TERRA recruitment to telomeres, a reporter system was developed for the study of ectopically-expressed TERRA-like RNA molecules. Employing this system revealed that TERRA can associate with chromosome ends post-transcriptionally in trans, in different phases of the cell cycle, forming R-loop structures preferentially at short telomeres.The study of the regulation of TERRA association with telomeres is of utmost relevance, given that the excess or scarcity of TERRA R-loops can impinge on telomeric stability and maintenance, thus potentially affecting cellular life span and tumorigenesis. Importantly, we show that TERRA R-loops are mediated by the DNA recombinase RAD51, which directly binds TERRA and stimulates TERRA invasion of telomeric DNA. On the other hand, we also report that the association of TERRA with telomeres through R-loops is counteracted by the THO complex (THOC) - a protein complex which links transcription with RNA processing and was previously shown to restrain the accumulation of DNA:RNA hybrids throughout the genome. We found that THOC counteracts R-loops formed at telomeres co-transcriptionally and also post-transcriptionally in trans - when TERRA is ectopically-expressed. We demonstrate that THOC binds nucleoplasmic TERRA, and that RNaseH1 loss increases THOC telomeric occupancy. Additionally, RAD51-mediated TERRA R-loop formation results in telomere fragility - which is indicative of defects in the semiconservative replication of telomeric DNA. Concurrently, THOC counteracts R-loop-derived telomeric fragility. Particularly, we found that THOC restrains telomeric fragility at telomeres replicated by lagging strand synthesis and mainly by leading strand synthesis. Finally, we observed that THOC suppresses telomeric sister chromatid exchange and C-circle accumulation in ALT cells, which employ a recombination-mediated telomere maintenance mechanism.Overall, this work contributes to the comprehensive characterization of the regulation of TERRA association with telomeres as R-loops, and explores the impact of the accumulation of these structures in telomere integrity.