Interdependent mode of RNA recognition by the interacting KH-domain proteins KhpA and KhpB

Abstract RNA-binding proteins (RBPs) are central regulators of gene expression. In bacteria, two KH domain-containing proteins, KhpA and KhpB, are increasingly recognized as globally-acting RBPs, including in pathogens that lack well-studied RBPs, such as Hfq and ProQ. While KhpA and KhpB interact with various RNAs in diverse bacteria, their crosstalk and molecular basis of RNA recognition remain poorly understood. Here, we explored the mutual dependence between KhpA and KhpB in the food-borne pathogen Campylobacter jejuni . We demonstrate KhpA and KhpB interact in vivo , and their individual global RNA recognition is largely dependent on the presence of the partner protein. Using RNA co-immunoprecipitation, we reveal that KhpA requires a conserved KH-domain motif for RNA binding, whereas KhpB relies on an atypical R3H domain. Moreover, mutating the RNA-binding domain of one protein compromises the RNA interactome of the other. Overall, we demonstrate an interdependent RNA-binding mode of KhpA and KhpB and anticipate that such KH-proteins have co-evolved to elicit joint RNA regulation in bacteria.