Nuclease escape elements protect messenger RNA against cleavage by multiple viral endonucleases

Mandy Muller; Britt A. Glaunsinger

SREs form a long stem-loop protruding structure with at least one bulge near the middle of 240 the stem, whereas the other regions of the SREs did not fold into any similar high-241 confidence structures (Fig. 4B) . To validate this predicted SRE stem loop structure 242 experimentally, we applied in-line probing, an RNA cleavage assay in which base-paired or 243 structurally constrained nucleotides are protected from spontaneous phosphodiester bond 244 hydrolysis [57] . Results from the cleavage reaction ( Fig. S4 ) largely confirmed the RNAfold 245 predictions, apart from a small variation in the IL-6 hairpin. We then tested whether this 246 structure is required for either IL-6 SRE or G-SRE function by changing two conserved TT 247 nucleotides located directly adjacent to the bulge in each hairpin structure to GG (SRE_GG; 248 mutated residues marked with asterisks Fig. 4B ). We also separately mutated the AA 249 residues predicted to base pair with these nucleotides on the other side of the loop to CC 250 (SRE_CC). Both the SRE_GG and the SRE_CC mutations in the IL-6 or GADD45B GFP fusions 251 resulted in partial degradation of these mRNAs upon SOX expression in 293T cells, 252