Innate immunity is a critical frontline host defense mechanism in response to pathogen infection.
At the onset of infections in vertebrates, a set of more than 400 genes are transcriptionally upregulated 51 by interferon and thus termed interferon-stimulated genes (ISGs). ISGs display diverse functions such 52 as activation of cell death programs and recruitment of immune cells (e.g. dendritic cells (Schneider et To investigate MISTRAV biology, we generated three A549 clonal cell lines -C15∆1, C15∆2, C15∆3 -182 with distinct indels that disrupted the MISTRAV ORF using CRISPR/CAS ( Figure 3A ). A549 cells were 183 selected because: 1) MISTRAV is interferon-inducible in these cells ( Figure 1D , Figure 
RT-PCR indicated that C15∆1 and C15∆2 cells lack full-length (FL) MISTRAV RNA expression in 192 IFN-g treated cells at steady-state while C15∆3 cells display a fortuitous and drastic increase of the same 193 transcript ( Figure 3C ). miRNA qPCR demonstrated that C15∆1 and C15∆2, maintain miR-147b at levels 194 comparable to wild-type with expression of miR-147b in C15∆3 ~30 fold greater than WT ( Figure 3D ).
Thus, C15∆1 and C15∆2 lack MISTRAV protein but maintain the miRNA, while C15∆3 lacks MISTRAV 196 with a gain-of-function of miR-147b expression.
Based on MISTRAV mitochondrial localization and numerous documented connections between 198 immune responses involving cell death mediated through mitochondria, we reasoned that MISTR might 199 mediate apoptotic responses. We primed WT and KO cells with IFN-g then added the commonly used 200 activator of apoptosis, staurosporine (STS), for 16 hours followed by functional analysis ( Figure 3E ).
Assays were normalized to either untreated controls or to the number of cells being tested to account for 202 differences in proliferation rates (Figure 3-figure supplement 1) . Interestingly, we observed that C15∆1 203 and C15∆2 displayed reduced sensitivity to STS, while C15∆3 showed increased sensitivity to STS 204 compared to WT cells ( Figure 3F ). Consistent with these results, we detected robust caspase-3/7 205 cleavage activity for C15∆3 compared to WT ( Figure 3G ; p<0.0001). In addition, detectable decreases in 206 caspase-3/7 activity were observed for C15∆1 and C15∆2 relative to WT treated cells ( Figure 3G ; 207 p<0.0001). The differential sensitivities of the clones to STS relative to WT were consistent with levels of 9 PARP cleavage across the clones in response to STS ( Figure 3H ). These data suggest a role for 209 MISTRAV and miR-147b in apoptosis. 
We define MISTRAV as an IFN-g-inducible gene ( Figure 1D ) and protein ( Figure proposed circuit had not been tested ( Figure 5H ).
Our model predicts that MISTR1 is a ubiquitously expressed sensor of stress. Specific stress 369 signals induce miRNA expression leading to the downregulation of MISTR1 and its replacement by 
The embedded nature of miR-147b implies a step-wise molecular progression of this response. 
The next day, cells were transfected with 50 ng/well of the psiCHECK-2 (Promega) construct using the 
Confocal Images. One day following transfection of either 1 µg hMISTRAV-GFP or 1 µg vMISTRAV-GFP,
HeLa cells were fixed and stained with mitoTracker Red (Thermo). Images were taken in the confocal 533 microscopy core at the University of Utah. 
The location of pre-mir-147b is marked by the blue box below predicted protein-coding mRNAs. 