Fig 2: Full-length NKRF containing the XTBD interacts with XRN2.(A) Schematic to show the three variants of NKRF that were expressed in U-2 OS cells. The expressed proteins are tagged at the N-terminus with a 3xFLAG tag. (B) U-2 OS cells were transiently transfected with the constructs expressing short, middle or full-length NKRF containing an N-terminal expressed FLAG tag. Cell extracts were generated and proteins were immunoprecipitated using anti-FLAG antibody. IgG was used as negative control. Isolated proteins were separated by SDS–PAGE and subjected to western analysis.

Fig 3: Actively transcribed nascent RNAs are bound to and degraded by Xrn2. (A) Diagram of 4-thio UTP NRO procedure. Pol II in isolated nuclei was ‘run on’ in the presence of 4thio UTP (star). Nascent, 4-thio UTP labelled, transcripts were purified by biotinylation (grey circle) and selection with streptavidin magnetic beads (black circle). These were then analysed by reverse transcription and real-time PCR. (B) Real-time PCR analysis of exon3-containing and spliced β-globin transcripts in nascent RNA samples obtained from mock or Xrn2 depleted cells transfected with WT, ΔTIn1m or ΔTpAm. Samples obtained from Xrn2 depleted cells were quantitated in relation to those obtained from control samples, which were set at 1 following normalisation to signal from co-transfected VA. (C) Analysis of Pol II loading on ΔTIn1m or ΔTpAm plasmids in mock and Xrn2 depleted cells using ChIP. Diagram shows positions of the PCR amplicons to detect Pol II loading at the promoter (grey arrows) and exon 3 (black arrows). Graph shows relative Pol II loading, calculated from the % input of each immunoprecipitation, which was given a value of 1 in control cells. (D) RNA immunoprecipitation analysis of Xrn2 bound transcripts in WT, ΔTIn1m and ΔTpAm transfected cells. Diagram shows positions of PCR amplicons to detect exon 3 (black arrows) and spliced (grey arrows) transcripts. Quantitation is expressed as fold enrichment over background expressed relative to that found for GAPDH mRNA, which was given a value of 1. All error bars represent the s.d. from a minimum of three biological replicates. *indicates P-value of <0.05.

Fig 4: Loss of XRN2 results in decreased tumor volume in vivo. (A) Magnetic resonance imaging was used to detect control (G55-shluc) and XRN2-deficient (G55-shXRN2) tumors in mouse brains. (B) Quantitation of tumor volumes obtained from control (G55-shluc) and two unique XRN2-deficient (G55-shXRN2 and G55-shXRN2 #2) G55 cell lines. * = p-value = 0.05, ** = p-value = 0.01. The Students t-test was used for statistical analysis.

Fig 5: Phosphorylation of Xrn2 occurs on chromatin. (A) Immunoblot analysis of soluble and chromatin-associated protein fractions. Soluble and insoluble proteins were loaded in equal amounts (left panel) or in fivefold excess of soluble protein (right panel). Pol II and a-tubulin were controls for insoluble and soluble fractions, respectively. (B) Immunoblot analysis performed as in A, (left panel) after 4 h of treatment with 1 µM flavopiridol (FP), 50 µM 5,6-dichloro-1-ß-D-ribofuranosyl-benzimidazole (DRB), or DMSO. Lamin B was used as a loading control for the insoluble protein fraction. (C) Depletion of Cdk9 causes loss of Xrn2 and Spt5 phosphorylation. Extracts of HCT116 cells infected with lentivirus expressing shRNA targeting CDK9 (shCdk9) or empty vector (shEmpty) were fractionated as in A and analyzed by immunoblotting for the indicated proteins.