R-loop, a three-stranded nucleic acid structure, has been recognized to play pivotal roles in critical physiological and pathological processes. Multiple technologies have been developed to profile R-loops genome-wide, but the existing data suffer from major discrepancies on determining genuine R-loop localization and its biological functions. Here, we experimentally and computationally evaluate eight representative R-loop mapping technologies, and reveal inherent biases and artifacts of individual technologies as key sources of discrepancies. Analyzing signals detected with different R-loop mapping strategies, we note that genuine R-loops predominately form at gene promoter regions, whereas most signals in gene body likely result from structured RNAs as part of repeat-containing transcripts. Interestingly, our analysis also uncovers two classes of R-loops: The first class consists of typical R-loops where the single-stranded DNA binding protein RPA binds both the template and non-template strands. By contrast, the second class appears independent of Pol II-mediated transcription and is characterized by RPA binding only in the template strand. These two different classes of RNA:DNA hybrids in the genome suggest distinct biochemical activities involved in their formation and regulation. In sum, our findings will guide future use of suitable technology for specific experimental purposes and the interpretation of R-loop functions.

Splicing factors are frequently mutated in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). These mutations are presumed to contribute to oncogenic transformation, but the underlying mechanisms remain incompletely understood. While no specific treatment option is available for MDS/AML patients with spliceosome mutations, novel targeting strategies are actively explored, leading to clinical trials of small molecule inhibitors that target the spliceosome, DNA damage response pathway, and immune response pathway. Here, we review recent progress in mechanistic understanding of splicing factor mutations promoting disease progression and summarize potential therapeutic strategies, which, if successful, would provide clinical benefit to patients carrying splicing factor mutations.