PACT-mediated regulation of endogenous dsRNA sensing by PKR

Protein kinase R (PKR), an innate immune sensor for double-stranded RNA (dsRNA), is critical for antiviral defense, but its aberrant activation by cellular dsRNA is linked to various diseases. The dsRNA-binding protein PACT plays a crucial and controversial role in the PKR pathway. We demonstrate that PACT is an essential negative regulator of PKR against endogenous dsRNA ligands like inverted repeat Alu RNAs, which satisfy PKR's selectivity for long dsRNA and robustly activate PKR in the absence of PACT. PACT employs an unusual inhibitory mechanism sensitive to dsRNA length and concentration, inhibiting PKR through low-affinity interactions most effective when both are bound to the same dsRNA molecule. Consequently, PACT's inhibition of PKR is more robust when dsRNA is longer and less abundant, with minimal effect on abundant or short dsRNA. Thus, PACT functions as a rheostat, setting the PKR activation threshold for long endogenous dsRNA without altering its inherent activity. Overall design: To identify the endogenous RNA ligands of PACT, we generated Flag-tagged PACT expressing HCC1806 cells. We then performed fCLIP with Anti-Flag antibody and sequenced the Input and CLIP samples. 2 biological replicates were prepared To identify the endogenous RNA ligands of PKR, we generated PKR/PACT double knock out cell lines in HCC1806 cells expressing HA-tagged PKR(K296R). We then performed fCLIP with Anti-HA antibody and sequenced the Input and CLIP samples. 3 biological replicates were prepared To identify the endogenous RNA ligands of PKR in PACT-sufficient and PACT-deificient backgrounds, we generated PKR knockout and PKR/PACT double knock out cell lines in HCC1806 cells expressing HA-tagged PKR(K296R). We then performed fCLIP with Anti-HA antibody and sequenced the Input and CLIP samples. 4 biological replicates were prepared