Fig 2: The interaction between NKRF and XRN2 is independent of rRNA.(A) U-2 OS cells were treated with the specific RNAPI inhibitor CX-5461 (CX) and cell extracts were generated from untreated and treated cells. Untreated cell lysates were incubated with either RNase or DNase for 2 h at 4°C. NKRF was then immunoprecipitated using an anti-NKRF antibody or the control IgG. The corresponding samples were separated by SDS–PAGE and immunoblotted with antibodies directed against NKRF and XRN2. Nucleolin was used as negative control. (B) U-2 OS cells were transfected with control siRNA (i) or siRNA targeting NKRF (ii) and after 72 h cells were fixed and permeabilised. XRN2 cellular distribution was determined by immunolocalisation. XRN2 is visualised in green; Hoechst (blue) and fibrillarin (red) were used as nuclear and nucleolar markers, respectively.

Fig 3: Full-length NKRF containing the XTBD restores the nucleolar localization of XRN2.(A) NKRF short, middle and full-length U-2 OS stable cell lines were transfected with an siRNA to the NKRF open reading frame (siORF), which would target both the endogenous and expression versions of the proteins, or the NKRF 3′UTR (si3′UTR), which would only target endogenous NKRF. After 72 h, the expression of the NKRF isoforms was induced with tetracycline for a further 24 h. Cell extracts were generated and immunoblotted with antibodies against either FLAG or NKRF. Endogenous NKRF is marked (red asterisk). (B) NKRF short (i), middle (ii) and full-length (iii) U-2 OS stable cell lines were transfected with siRNAs targeting the 3′UTR of endogenous NKRF. After 72 h, expression of the FLAG-tagged versions of NKRF was induced for 24 h with tetracycline. Cells were fixed and permeabilised and the subcellular distribution of XRN2 (red) was determined using immunolocalization. NKRF (green) was visualised using anti-FLAG antibodies. Nuclei were stained with Hoechst (blue). (C) The data in (B) were quantified by counting ∼100 cells per condition to assess the XRN2 distribution in the nucleoli after expression of the different version of NKRF. The percentage of cells where XRN2 was present in the nucleoli was calculated (n = 3, error bars represent SD, and the significance was calculated using Fisher's exact test).

Fig 4: 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 5: Full-length NKRF contains an XRN2 binding domain (XTBD).(A) Cell extracts from HeLa, U-2 OS, A549, MCF7, RAW264.7, HT29, SH-SY5Y, Jurkat and HEK293 cells were separated by SDS PAGE and subjected to western analysis using an antibody against NKRF. Actin was used as a loading control. (B) NKRF was immunoprecipitated and subjected to mass spectrometry which identified full-length NKRF (NKRF_full) containing an intact XTBD domain. The sequence of the N-terminus of the extended NKRF is depicted with the sequence that is unique to NKRF_full in the grey dotted box. The three sequences that are unique to NKRF_full that were identified by mass spectrometry are highlighted in grey. Peptides that were identified in these experiments are represented as blue lines. (C) A schematic presentation of NKRF proteins is shown representing (i) the short isoform of 690 amino acids, (ii) the middle isoform of 705 amino acids, and (iii) the full-length protein of 784 amino acids. All isoforms of NKRF contain three RNA binding domains: G-Patch, RH3, and double strand RNA binding domain. Only full-length NKRF contains the intact XRN2 binding domain (red). (D) The NKRF XRN2 binding domain (XTBD) contains functionally important conserved residues. Sequence comparison of XTBD from CDKN2AIP (from mouse, rat and human), C2AIL (CDKN2AIPNL) (from mouse, rat, bovine and human), PAXT-1 (C. elegans) and human full-length NKRF shows a high degree of conservation and particularly residues Cys54 and Tyr56 (orange), which are essential for the interaction with XRN2. (E) A structural model of the NKRF XTBD domain was generated using RaptorX [32] and this structure was superimposed on the crystal structure of PAXT-1 using PyMol. The residues Cys54 and Tyr56, which are essential for the interaction with XRN2, are shown in orange.