Bacterial Riboswitches and Ribozymes Potently Activate the Human Innate Immune Sensor PKR

The
innate immune system provides the first line of defense against
pathogens through the recognition of nonspecific patterns in RNA to
protect the cell in a generalized way. The human RNA-activated protein
kinase, PKR, is a dsRNA binding protein and an essential sensor in
the innate immune response, which recognizes viral and bacterial pathogens
through their RNAs. Upon activation via RNA-dependent
autophosphorylation, PKR phosphorylates the eukaryotic initiation
factor eIF2α, leading to termination of translation. PKR has
a well-characterized role in recognizing viral RNA, where it binds
long stretches of double-stranded RNA nonsequence specifically to
promote activation; however, the mechanism by which bacterial RNA
activates PKR and the mode by which self RNA avoids activating PKR
are unknown. We characterized activation of PKR by three functional
bacterial RNAs with pseudoknots and extensive tertiary structure:
the cyclic di-GMP riboswitch, the glmS riboswitch-ribozyme,
and the twister ribozyme, two of which are ligand-activated. These
RNAs were found to activate PKR with comparable potency to long dsRNA.
Enzymatic structure mapping in the absence and presence of PKR reveals
a clear PKR footprint and provides a structural basis for how these
bacterial RNAs activate PKR. In the case of the cyclic di-GMP riboswitch
and the glmS riboswitch-ribozyme, PKR appears to
dimerize on the peripheral double-stranded regions of the native RNA
tertiary structure. Overall, these results provide new insights into
how PKR acts as an innate immune signaling protein for the presence
of bacteria and suggest a reason for the apparent absence of protein-free
riboswitches and ribozymes in the human genome.