Amino acids 26 to 51 of the mouse RNase L are required for interaction with and inhibition by L*.

<p>A. The structure of the ankyrin domain of tested mouse/human RNase L chimeras is presented under a cartoon showing the general organization of RNase L. Blue thick lines (with numbered residues) represent the RNase L segments within the ankyrin domain (to scale with the upper cartoon) that were exchanged between human and mouse RNase L. The table given right of the cartoon summarizes binding and functional data obtained with symmetrical mouse and human RNase L chimeras. B-C. Co-immunoprecipitation of indicated Flag-RNase L (left: mouse RNase L, right: human RNase L) with HA-L*<sub>DA</sub>. Immunoblots (B) show Flag and HA detection after immunoprecipitation of HA-L* (IP:HA) and in cell lysates (Input). Graphs (C) show the mean and SD of the amount of co-immunoprecipitated RNase L chimera relative to that of WT mouse RNase L (n = 3). *: p<0.05 in a two-way ANOVA followed by Dunnett’s test for multiple comparison. D. Analysis of RNase L-mediated RNA degradation in HeLa-M cells overexpressing indicated Flag-RNase L and L*<sub>DA</sub>. RNA samples were extracted 7 hours after polyI:C transfection. Arrowheads point to rRNA cleavage products. Reproducible results were obtained in 2 independent experiments. E. Alignment of rat, mouse and human RNase L protein sequences. The upper part shows a schematic representation of the alignment. Blue lines represent amino acids that differ from the two other sequences. Percentages of sequence identity are indicated on the right of the alignment. The lower part shows a zoom in ANK R1 and R2 amino acid sequences. Blue residues are residues that differ from the two other sequences. Asterisks indicate amino acids that were tested in the chimeric constructs. Underlined sequences correspond to those affecting L* binding.</p>