Live-cell imaging unveils distinct R-loop populations with heterogeneous dynamics.

We have developed RHINO, a genetically encoded sensor that selectively binds RNA:DNA hybrids enabling live-cell imaging of cellular R-loops. RHINO comprises a tandem array of three copies of the RNA:DNA hybrid binding domain of human RNase H1 connected by optimized linker segments and fused to a fluorescent protein. This tool allows the measurement of R-loop abundance and dynamics in live cells with high specificity and sensitivity. Using RHINO, we provide a kinetic framework for R-loops at nucleoli, telomeres and protein-coding genes. Our findings demonstrate that R-loop dynamics vary significantly across these regions, potentially reflecting the distinct roles R-loops play in different chromosomal contexts. RHINO is a powerful tool for investigating the role of R-loops in cellular processes and their contribution to disease development and progression.

Live-Cell Imaging of Transcriptional Activity at DNA Double-Strand Breaks.

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far elusive how DSBs affect transcription. A reason for this was the lack of suitable tools to simultaneously monitor transcription and the induction of a genic DSB with sufficient temporal and spatial resolution. This work describes a set of new reporters that directly visualize transcription in live cells immediately after the induction of a DSB in the DNA template. Bacteriophage RNA stem-loops are employed to monitor the transcription with single-molecule sensitivity. For targetting the DSB to a specific gene region, the reporter genes are engineered to contain a single recognition sequence of the homing endonuclease I-SceI, otherwise absent from the human genome. A single copy of each reporter gene was integrated into the genome of human cell lines. This experimental system allows the detection of single RNA molecules generated by the canonical gene transcription or by DNA break-induced transcription initiation. These reporters provide an unprecedented opportunity for interpreting the reciprocal interactions between transcription and DNA damage and to disclose hitherto unappreciated aspects of DNA break-induced transcription.