FIG. 3.
Msi1 protein binds to the 3′-UTR of numb mRNA in vitro. (A) Structure of the numb gene according to a previous report (70). Arrows (N1, N2, and N3) indicate the regions that were individually transcribed in vitro. The vertical arrowhead indicates the region containing a putative Msi1-binding sequence (UAGGUAGUAGUUUUA). (B) Binding assays of the Msi1 protein to various transcripts from the 3′-UTR of m-numb mRNA. 35S-labeled full-length Msi1 protein (F) was pulled down with biotin-labeled N2 m-numb RNA-conjugated streptavidin-agarose beads and then the protein-RNA interaction was visualized by autoradiography. The other portions of m-numb RNA, N1 and N3, were not significantly bound by Msi1 protein. In lane −, the absence of biotin-labeled RNA never caused coprecipitation of Msi1. The rectangle on the right shows the amount of total protein included per assay. (C) Schematic representation of Msi1 fusion proteins. F, full-length mouse Msi1; R, truncated protein containing two tandem RBDs (Msi1-2TR); C, truncated protein containing C-terminal potion of mouse Msi1. (D) Both full-length Msi1 protein (F) and truncated Msi1-2TR protein (R, used for SELEX) bind N2 RNA which contains the selected Msi1-binding sequence. The truncated protein containing two RNA-binding domains (R) conserves RNA-recognition specificity similar to that of the full-length Msi1 protein. The C-terminal half of Msi1 protein (C) did not bind N2 RNA. (E) The putative Msi1 binding site in 3′-UTR of m-numb mRNA is located in the loop portion of a predicted stem-loop structure. The predictions of RNA secondary structure were based on the Zuker-Stiegler method.