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Fig. 1. Characterization of the HOW response element. Western blot analysis of HA-tagged HOW following precipitation with distinct biotin-labeled RNA fragments. (A) Top, schematic representation of the putative HOW response element (HRE) sites in the stripe 3'UTR. Bottom, the sequences of the HRE sites in the RNA samples (at 0.4 µM concentration) are indicated below the blot; wild type (ACUAA, left) or with point mutations in the HRE site (AC, middle or AG, right). HOW^m was used as a control for non-specific binding because it mimics the how^e44 allele, which does not bind RNA. (B) The wild-type stripe 3'UTR (0.4 µM) (two right-hand lanes) or the stripe 3'UTR with point mutations in all four HRE sites (two left-hand lanes). (C) Binding of HOW to RNA fragments (0.4 µM) representing 0-533 nucleotides of the stripe 3'UTR (0 represents the last nucleotide of the stop codon), containing a single HRE site (1), or representing nucleotides 0-610, containing three HRE sites (3). (D) HRE in loop structures of variable sizes (loop sizes are indicated) fused to the 1-225 nucleotide (nt) RNA fragment of stripe 3'UTR (0.4 µM). (E) Biotin-labeled RNA oligomers containing a single HRE within the distinct secondary-structure motifs; unstructured, loop, stem, stem and loop junction 1, and stem and loop junction 2 (illustrated below) at concentrations of 0.2 µM (1), 0.02 µM (2), 0.002 µM (3). ^*Indicates the HRE.

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