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

Mandy Muller; Britt A. Glaunsinger

To further validate the interactions with NCL, HuR, and hnRNPU, we 288 immunoprecipited (IP) each endogenous protein from 293T cells and performed RT-qPCR 289 to measure the level of co-precipitating endogenous GADD45B RNA. We observed a >5-290 fold enrichment of GADD45B mRNA over the mock (IgG) IP for both NCL and HuR, although 291 we were not able to detect an association with hnRNPU in this assay (data not shown) (Fig. 292 5D ). We confirmed that the interaction occurs on the GADD45B 3'UTR by performing the 293 IPs from cells transfected with a GFP reporter fused to either the GADD45B 5' UTR or 3' 294 UTR (Fig. 5E) . The structurally compromised mutant GADD45B 3'UTR_GG described in Fig. 295 4B failed to interact with NCL and HuR, confirming that the stem-loop structure in the SRE 296 is important for protein binding (Fig. 5E) . Collectively, these data suggest that while there 297 is some overlap between the sequence, structure, and RNA binding proteins associated 298 with the GADD45B and IL-6 SREs, these elements likely assemble distinct RNP complexes 299 and thus may function in a related but non-identical manner. 300 301 302