DNA-RNA three-stranded hybrids: from the telomere to the genome (mouse dataset)

The origin and possible functions of local three-stranded DNA-RNA hybrid regions in the mammalian genome is still a matter of discussion. R-loops have been so far essentially detected by immunodetection of the DNA-RNA hybrid regions (DRIP assay). This powerful and easy to implement method relies on recognition by a unique monoclonal antibody and may not detect all the hybrid regions. Starting from simple logical steps, we developed a preparative method that allows detection and physical isolation of stable DNA-RNA triplexes in a complex genome. A modification of the popular TRIzol extraction procedure first identifies RNAs stably bound to DNA in RNaseH-sensitive complexes, which can be isolated and further characterized by RNA-seq. We first tested the efficiency of the approach by a search for a generalized three-stranded state of the human and mouse telomeres. While such a structure is suggested by the complementarity of the long chromosomal CCCTAA repeats and the noncoding RNA TERRA, it was only in one class of cancer cells that telomeric R-loops were so far evidenced by DRIP assay. The proposed method allowed us to generalize to the mouse and human genomes the notion of terminal R-loop complexes with TERRA molecules synthesized from local promoters. When further applied to murine sperm genome, it evidenced a reproducible genome-wide profile of RNA complexes. Overall design: Examination of DNA/RNA hybrids and free-RNAs in sperm and testis