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

Mandy Muller; Britt A. Glaunsinger

Recently, we showed that KSHV SOX cleaves its targets at a specific but degenerate 215 RNA motif [21] . Thus, the failure of SOX (or perhaps vhs) to degrade the GADD45B mRNA 216 could either be due to the absence of such a targeting motif (e.g. passive escape), or to the 217 presence of a specific protective element like the IL-6 SRE (e.g. dominant escape). To 218 distinguish these possibilities, we constructed chimeras between GFP, which has a well-219 characterized SOX cleavage element, and the GADD45B 5' UTR, 3' UTR, or coding region 220 (CDS), each of which were cloned downstream of the GFP coding region (Fig. 3D) . Co-221 transfection of the GFP-fused GADD45B 3' UTR construct with SOX into 293T cells did not 222 lead to degradation of this mRNA, whereas the GFP-GADD45B 5' UTR or CDS fusions were 223 readily degraded in SOX-expressing cells (Fig. 3E) . Thus, similar to the IL-6 3' UTR, the 224 To refine which GADD45B sequence encompassed the SRE, we initially looked for 230 similarities between the GADD45B 3' UTR and the IL-6 SRE using Clustal W alignment. 231