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

Mandy Muller; Britt A. Glaunsinger

including the SOX homologs [1, 9, 21, 40, 41] . Thus, the IL-6 derived SRE is unlikely to 144 function through steric occlusion of a common cleavage site. We instead considered the 145 possibility that the SRE functions like an RNA 'zip code', directing its associated transcript 146 to a location in the cell inaccessible to endonucleases. To test this hypothesis, we used RNA 147 fluorescence in situ hybridization (FISH) to monitor how the presence of the IL-6 SRE 148 impacted the localization of its associated mRNA. Six phage MS2-derived stem-loops were 149 introduced upstream of the SRE or ΔSRE segment of the IL-6 3' UTR in the pcDNA3 150 luciferase reporter, enabling visualization of the RNA in transfected 293T cells using a Cy3-151 labeled RNA probe directed against the MS2 sequences [42] . We verified that the presence 152 of the stem-loops did not prevent the escape of the SRE-containing reporter or degradation 153 of the ΔSRE reporter in SOX-expressing cells (Fig. S1) . There was no distinguishable 154 difference in the localization of the SRE and ΔSRE containing mRNAs, both of which were 155 present relatively diffusely throughout the cell (Fig. 1C) . The FISH signal was specific to 156 transfected cells, as we observed no fluorescence in neighboring untransfected cells nor 157 autofluorescence from transfected cells lacking the Cy3 probes (Fig. 1C) . Although these 158 data to not exclude the possibility that the SRE-containing transcript became sequestered 159 into micro-aggregates or other structures not visible at this level of resolution, they do not 160 support relocalization as the driver of escape from viral endonuclease cleavage. 161 162 We next considered whether the inability to cleave an IL-6 SRE-containing 163 transcript was specific to viral endonucleases or similarly extended to host endonucleases. 164