5070 Nucleic Acids Research, 2012, Vol. 40, No. 11 Stem–loop I of IBV 50-UTR is required for interaction with MADP1 To deﬁne the segment and structural elements of IBV 50-UTR required for its interaction with MADP1, four truncated mutant RNA fragments were synthesized, as shown in Figure 3A, by in vitro transcription. 50-UTR 1 contains stem–loops I–IV (38), 50-UTR 2 and 50-UTR 3 spans stem–loops I to III and II to IV, respectively, whereas 50-UTR 4 spans the rest of the 388 nucleotides. The biotin-labeled RNA transcripts were used in the biotin-RNA pull-down assay (Figure 3B) to check the efﬁciency of Flag-tagged MADP1 co-puriﬁcation with RNA. Results showed that MADP1 was co-puriﬁed only with transcripts which contain stem–loops I–III of the 50-UTR (50-UTR 1 and 50-UTR 2). In addition, stem–loop I appeared to be essential for interacting with MADP1 as its absence in 50-UTR 3 abolished the inter- action with MADP1 (Figure 3B). The rest region of the 50-UTR (50-UTR 4) did not appear to interact with MADP1 (Figure 3B). To conﬁrm further the role of stem–loop I in the inter- action between MADP1 and IBV 50-UTR, two mutants were constructed, based on 50-UTR 2. 50-UTR 2M1 carried two-point mutations at nucleotide residues 11 and 12 from GA to CU, which would disrupt the structure of stem–loop I (Figure 3C), and 50-UTR 2M2 carried additional mutations at residues 25 and 26 from UC to AG (Figure 3C), which would restore the secondary struc- ture of stem–loop I. The mutant RNAs spanning stem– loops I–III were assessed for its ability to bind MADP1. The result indicated that the integrity of stem–loop I may be essential for the interaction between the 50-UTR with MADP1 (Figure 3D), as the stem–loop disrupting mutation (50-UTR 2M1) failed to interact with MADP1. The stem–loop restoring mutation at nucleotide residues 25 and 26 from UC to AG was able to restore partially the interaction (50-UTR 2M2) (Figure 3D). This result afﬁrmed the conclusion that the secondary structure of stem–loop I of IBV 50-UTR is indispensable for its interaction with MADP1. The RNA recognition motif (RRM) of MADP1 is responsible for its interaction with IBV 50-UTR MADP1 contains two nucleic acid binding domains, the RNA recognition motif (RRM) in the N-terminal region and the universal minicircle sequence binding protein (UMSBP) in the central region. In order to identify the domain involved in the interaction between MADP1 and IBV 50-UTR, a series of truncation mutants of the protein were created (Figure 4A). The ﬁrst three mutants, Madp1n which contains the RRM domain, Madp1m spans the zinc ﬁnger domain and Madp1c contains mostly phosphorylation sites, were assessed for their ability to interact with IBV 50-UTR. Only Madp1n Figure 3. Deﬁning the protein interaction sequence. (A) A schematic diagram of the RNA probes used to deﬁne the interaction region. Numbers denote nucleic acid residue position and roman numerals denote stem–loop number. The boundary of the leader sequence of IBV (nt. 1–64) is marked by the box on the full-length 50-UTR. (B) Interaction of various deletion constructs of IBV 50-UTR with MADP1. Total cell lysates prepared from H1299 cells over-expressing Flag-tagged MADP1 were mixed with RNA probes spanning different regions of IBV 50-UTR. The RNA–protein complexes were puriﬁed with streptavidin beads, resolved by SDS–PAGE and probed with antibody to Flag tag for the presence of Flag-tagged MADP1 protein. (C) Diagram showing the two mutants of 50-UTR 2 containing either two point mutations which disrupt stem–loop I (50-UTR 2M1) or a mutant restoring stem–loop I in 50-UTR 2M1 (50-UTR 2M2). (D) The essential role of a stem–loop I in the interaction between MADP1 and IBV 50-UTR. Total cell lysates prepared from H1299 cells over-expressing Flag-tagged MADP1 were mixed with 50-UTR 2M1 and 50-UTR 2M2, respectively. The RNA–protein complexes were puriﬁed with streptavidin beads, resolved by SDS–PAGE and probed with antibody to Flag tag for the presence of Flag-tagged MADP1 protein.