Nascent RNA Occupancy Profiling

NRO was conducted as previously described (10 (link),11 (link),38 (link)) and used to measure the transcription activities of the MT genes upon MRE circRNA overexpression. In short, after subcellular fractionation, nascent transcripts in nuclei were labeled with bromouridine triphosphate (BrUTP, Sigma, B7166) for 5 min in NRO Buffer (50 mM Tris–HCl pH 7.5, 5 mM MgCl₂, 150 mM KCl, 0.1% (w/v) srkosyl, 10 mM dithiothreitol and 80 units/ml RNase inhibitor (Beyotime, R0102)), isolated by anti-BrUTP (Abcam, ab1893) following the standard immunoprecipitation procedure and subjected to RT-qPCR assays. Purity of nascent RNAs was confirmed by checking the relative pre-mRNA enrichment of MtnA and MtnB in NRO samples over that in whole-cell RNA extracts.