R-loops do not accumulate in transcription-defective hpr1-101 mutants: implications for the functional role of THO/TREX

To get further insight into the effect that THO/TREX and R-loops have in transcription-associated recombination and transcription, we analyzed the ability to form R-loops of hpr1-101, a THO mutation that impairs transcription and mRNP biogenesis without triggering hyper-recombination. Human AID, a cytidine deaminase that acts on ssDNA displaced by RNA-DNA hybrids, strongly induced both hyper-recombination and hyper-mutation in hpr1-101, similar to hpr1Delta mutants. However, in contrast to hpr1Delta, AID-induced mutations in hpr1-101 occur at similar frequencies in both the transcribed and non-transcribed strands, implying that the enhanced AID action in these mutants is not caused by co-transcriptional R-loops. These results indicate for the first time that THO has a transcriptional function that is not mediated by R-loops, providing a new perspective for the understanding of the coupling of transcription with mRNP biogenesis and export.