Figure 3: Biochemical and biophysical properties of the cap-binding region of NCBP3.

(a) Top, predicted features of NCBP3 and their positions in the full-length protein: RRM fold (aa 126–187, highlighted in blue), nuclear localization signal (NLS) and glutamine-rich region (Glu-rich). Bottom, secondary structure elements of NCBP3 (aa 120–189) and sequence alignment with human and mouse PARN RRMs. Arrowheads show residues chosen for a mutational analysis. (b) Homology model of the RRM of NCBP3. Secondary structure elements of the predicted RRM fold and residues chosen for mutational analysis are annotated. (c) Modelled RRM domain of NCBP3 superimposed with mouse PARN bound to m7GpppG (for clarity only the m⁷Gp moiety is shown, PDB code 3d45) to indicate the putative binding site. The model shows the solvent-accessible surface coloured according to electrostatic charge distribution. (d) Binding of E. coli-expressed NCBP3 to RNA oligonucleotides. Western blot after affinity purification with 5′-CAP- or OH-RNA using lysate from bacteria expressing recombinant full-length NCBP3. Full-length (fl.) recombinant NCBP3 and N-terminal degradation products (deg.) are indicated. (e) m⁷GTP versus GTP-affinity purification and western blotting of recombinant NCBP3 from E. coli lysates. (f) Coomassie-stained SDS–polyacrylamide gel electrophoresis gel after affinity purification of purified wild-type and mutant (ALAA) NCBP3 (aa 1–282) using m⁷GTP and GTP as bait. (g) Binding affinity of NCBP3 (aa 1–282) to 5′-CAP- or 5′-OH-RNA as determined by microscale thermophoresis (MST). The graph shows normalized fluorescence versus concentration of NCBP3. Mean±s.d. of three independent measurements are shown. The inset shows the quality of the recombinant NCBP3 (aa 1–282) as analysed by an Agilent protein chip. (h) Binding of recombinant wild-type and mutant NCBP3 to 5′-capped RNA. Western blot after affinity purification with biotinylated RNA oligos harbouring either a 5′-CAP or OH structure using lysate from E. coli expressing either recombinant full-length wild-type NCBP3 (wt), NCBP3 where aspartic acid at position 134 had been mutated to alanine (D134A), or where tryptophan at position 155 and two aspartic acids at position 157 and 158, respectively, had been mutated to alanines (ALAA).