CRAC of Nab3 in yeast cells accumulating a circular RNA containing Nab3 binding sites (the decoy) or an empty control

We report the analysis of in vivo, genomewide binding of Nab3 under conditions in which non-degradable targets of Nab3 accumulate. We aimed at mimicking the effect of defective degradation of non-coding RNAs (Cryptic Unstable Transcripts, CUT) that can be bound by Nab3 on the co-transcriptional recruitment of Nab3 for transcription termination. The RNA was crosslinked to Nab3 by exposing the cells to UV irradiation and sequenced after purification of HTP-tagged Nab3 under stringent and denaturing conditions. This reveals the extent of binding of Nab3 to its different targets . Circular RNAs were generated by preventing debranching and subsequent degradation of an intron lariat containing a CUT in a strain deleted for the debranching enzyme Dbr1 as described in Villa, T., Barucco, M., Martin-Niclos, M.-J., Jacquier, A. & Libri, D. Degradation of Non-coding RNAs Promotes Recycling of Termination Factors at Sites of Transcription. Cell Reports 32, 107942 (2020).