retained a low level of the RNA-binding activity (Figure 4B, 1n) and negligible activity was detected for the other two truncated proteins (Figure 4B, 1m, 1c). As the RNA-binding activity for Madp1n fragment was much lower compared to the full-length protein, three more mutants were created to extend the Madp1n fragment (Figure 4A). An extension of 14 or 31 amino acid residues was made for mutants Madp1x and Madp1z, respectively. A truncation at the N-terminus by 40 residues as well as an extension by 14 amino acid residues was made for Madp1y. It was observed that both Madp1x and Madp1z bound to IBV 50-UTR more strongly than did the full-length protein as well as Madp1n mutant protein (Figure 4B, 1x, 1z). Madp1y, on the other hand, bound weakly to the RNA fragment (Figure 4B, 1y). Hence, the 14 amino acid extension beyond the RRM (Madp1x) may have been required to preserve the integrity of the protein structure and that the Nucleic Acids Research, 2012, Vol. 40, No. 11 5071 40 amino acid residues at the N-terminus of MADP1 are required for efﬁcient RNA binding. As the RRM domain was determined to be responsible for the interaction, information available on this domain indicated three amino acids at its active site, which interact with nucleic acid residues via their aromatic and hydro- phobic side chains. For MADP1, the identiﬁed active site was composed of phenylalanine 55 and valine 53, respect- ively, while tyrosine 13 may have acted as an anchor for the phosphate backbone via electrostatic interactions. Hence, three mutants with either a single alanine substi- tution for tyrosine 13 (Y13A), a double alanine substitu- tion for valine 53 and phenylalanine 55 (V53F55A) or triple alanine substitutions for all three residues (YVF), were constructed (Figure 4A). These three mutants were over-expressed in H1299 cells as Flag-tagged proteins, and the lysates were assessed for their respective RNA-binding afﬁnities for full-length IBV 50-UTR (Figure 4C). Figure 4. Deﬁning the RNA-binding domain. (A) A schematic diagram of constructs of MADP1 and its truncation mutants. Numbers denote amino acid residue positions. Conserved domains RNA Recognition Motif (RRM) and Universal Minicircle Sequence Binding Protein (UMSBP) were indicated as black and white blocks, respectively. (B) Interaction of deletion mutants of MADP1 with IBV 50-UTR. Cell lysates prepared from H1299 cells over-expressing Flag-tagged wild-type MADP1 or its truncation mutants were used for biotin-RNA pull-down assay using the full-length IBV 50-UTR. Both the crude lysates (labeled C) and protein bound on the streptavidin beads (labeled E) were resolved by SDS–PAGE and detected by Western blot with anti-Flag antibody. EGFP over-expressed cell lysate was included as a negative control. (C) Interaction of three MADP1 mutant constructs, Y13A, V53F55A and YVF, with IBV 50-UTR. The three full-length MADP1 constructs with amino acid mutations at the predicted RNA-binding sites were transfected into H1299 cells and used in a biotin-RNA pull-down assay with the full-length IBV 50-UTR.