Fig. 4: The catalytic activity of Xrn2 is indispensable for its function.

a Degradation of nascent RNA by Xrn2 but not RNase I_f dislodges Pol II. In vitro transcription termination assay was performed using Pol II immobilised on beads via biotinylated non-template DNA (schematic in Supplementary Fig. 4f). S - corresponds to the supernatant fraction, B – beads (n = 3, termination assessed by Western Blot). Source data are provided as a Source Data file. b Auxin-mediated depletion of Xrn2. Additional strains were created where WT or catalytically inactive Xrn2 mutant (Xrn2^M - D235A) were introduced to complement depleted protein. Western blot analyses of AID-Flag tagged Xrn2 and constitutively expressing V5-tagged WT or Xrn2^M. Histone (H3) serves as a loading control. Source data are provided as a Source Data file. c Assessment of transcription termination using endogenous phosphatase activity-based reporter system (Supplementary Fig. 5d). Phosphatase activity of secreted acid phosphatase Pho1 serves as a proxy for termination efficiency. Xrn2^M cannot complement the depletion of the WT enzyme. Data are presented as mean values +/− SEM (n = 4). Statistical analysis is included in Supplementary Data 5. Source data are provided as a Source Data file. d Functional analyses of transcription upon acute depletion of Xrn2 or expression of catalytically inactive Xrn2 by transient transcriptome analysis (TT-seq). Representative snapshots demonstrating severe readthrough transcription in the absence of Xrn2 or expression of catalytically inactive Xrn2 (full signal Supplementary Fig. 5f). e Heatmap showing gene clusters differentially affected upon Xrn2 depletion or inactivation. Transcription on sense and anti-sense strands is presented as a log₂ fold ratio to control strain (DMSO treated strain without Xrn2 complementation). Cluster 1 and cluster 2 indicate that transcription downregulation correlates with readthrough on the opposite strand. Cluster 2 includes genes that appear as upregulated. Coding TUs that do not have another gene on the same strand within 250 bp upstream of TSS or downstream of PAS were selected for analysis (n = 3190)^146,149. f Evaluation of genes in cluster 2 (Fig. 4e) presented as relative metagene. Accumulation of signal before TSS indicates that upregulation is caused by global readthrough. g Loss of Xrn2 activity results in transcription termination defects at Pol I transcribed rRNA genes.