(A) Diagram of Renilla Luciferase (RnLuc) reporters including the hb 3´UTR. WT PRE1 and PRE2 sequences, located within NRE1 and NRE2, respectively, and mutant PRE sequences (mt1, mt2, and mt1,2) are shown. (B) Diagram of Nos protein. Amino acid residue boundaries of the N-terminal region (N), central region (Z, blue) including ZFs, and C-terminal extension (C) are indicated. (C) Nos-enhanced repression via the hb PREs. Reporter assays were performed in D.mel-2 cells. Percent repression values are graphed for RnLuc WT and mt hb 3´UTR reporter expression with negative control Halo-tag alone (Halo) and full length Halo-Nos test proteins. (D) The Nos Z and C-terminal regions together retain efficient repression activity. Percent repression values are graphed for RnLuc hb 3´UTR WT reporter expression with negative control Halo and Halo-Nos variants are shown. For panels C and D, mean and standard error of the mean (SEM) values from quadruplicate experiments are shown. Expression of test proteins was visualized by tetramethylrhodamine (TMR) fluorescent labeling of the Halo-tag fusion proteins. Statistical analysis of the data is reported in Figure 1―source data 1.