5068 Nucleic Acids Research, 2012, Vol. 40, No. 11 anti-sense 30-UTR, which was not required for viral protein translation, subsequent functional studies of the protein would prove to be complicated as the virus itself relies heavily on the host ribosome to translate viral proteins, necessary for the infection to proceed. Therefore, it was not chosen for further studies. HAX1 was reported to function as an anti-apoptotic protein, which was not the focus of our screen and was therefore not chosen for further studies as well. MADP1 was reported as a member of the alternative splicing pathway, which implied a possible role in facilitating distal RNA sequences to be brought into close proximity, corresponded well with current evidence on the mechan- ism of discontinuous transcription. Therefore, it was chosen as the sole target for this study. The 50-UTR of coronavirus genomic RNA interacts speciﬁcally with MADP1 The interaction between MADP1 and the coronavirus 50-UTR was conﬁrmed by using over-expressed Flag-tagged MADP1 in a biotin-RNA pull-down assay. Based on the efﬁciency of Flag-tagged MADP1 co-puriﬁcation with the biotinylated RNA, the full-length, mammalian-expressed MADP1 was found to be able to interact with the 50-UTR of IBV and SARS-CoV RNA (Figure 1A). Over-expressed Flag-tagged protein was used to facilitate detection, as there was no commercially avail- able antibody to the protein at that time. It was noted that IBV 50-UTR showed higher binding afﬁnity to the Flag-tagged MADP1 than did SARS-CoV 50-UTR (Figure 1A). The speciﬁc interaction between IBV 50-UTR and MADP1 and its functional implication in coronavirus replication were therefore chosen for subse- quent characterization. To check the speciﬁcity of the interaction, a competition assay based on the biotin-RNA pull-down assay was per- formed. Total cell lysates containing Flag-tagged MADP1 were incubated with 0.1 mM biotinylated IBV 50-UTR in the presence of increasing concentrations of either un- labeled speciﬁc competitor RNA probe (IBV 50-UTR) or unlabeled non-speciﬁc probe (EGFP RNA) composed of nucleotides 1–528 of the EGFP coding sequence, from 0 to 0.2 mM. Western blot analysis of the co-puriﬁed Figure 1. MADP1 interacts speciﬁcally with IBV 50-UTR.(A) Interaction of MADP1 with SARS-CoV and IBV 50-UTR in a biotin-RNA pull-down assay. Total cell lysates prepared from H1299 cells over-expressing Flag-tagged MADP1 were mixed with 0.1 mM of biotinylated IBV and SARS-CoV 50-UTR, respectively, followed by addition of streptavidin agarose beads. Unbound complexes to the beads were subsequently removed by washing and complexes that remained bound to the beads were eluted with gel loading buffer. All fractions and elute were resolved by SDS–PAGE and probed with antibody to Flag tag. (B) Competition assay for the speciﬁcity of interaction between MADP1 and IBV 50-UTR. Total cell lysates prepared from H1299 cells over-expressing Flag-tagged MADP1 was added to mixtures of 0.1 mM biotinylated IBV 50-UTR RNA and varying concentrations of unlabeled IBV 50-UTR or EGFP RNA. Streptavidin agarose beads were added and treated under conditions identical to (A). Total cell lysates prepared from cells over-expressing Flag-tagged IBV N protein were added to mixtures of a ﬁxed concentration of biotinylated IBV 30-UTR RNA and unlabeled IBV 30-UTR or EGFP RNA and subjected to the same treatment.