RNA–RNA interactome approaches provide <i>in vivo</i> evidence for a critical role of the Hfq rim face in sRNA–mRNA pairing

Most bacterial small regulatory RNAs (sRNAs) modulate gene expression by forming complementary base pairs with target messenger RNAs (mRNAs), dependent upon the RNA chaperone Hfq. Hfq has three RNA-binding faces (proximal, rim, and distal), facilitating simultaneous binding of sRNAs and target mRNAs. Here, we systematically examined the functional impact of amino acid substitutions in the RNA-binding faces on in vivo pairing, using the RNA interaction by ligation and sequencing (RIL-seq) approach. The distal and proximal Hfq-binding face mutants retained substantial numbers of RNA-RNA interactions (significant chimera counts or S-chimeras). However, the rim face mutant R16A showed a near-complete loss of S-chimeras, although Hfq R16A retained partial RNA-binding activity as well as partial regulatory activity. Intracellular RIL-seq, a method with fewer in vitro processing steps, led to more S-chimeras in R16A, but there were still fewer than for wild-type Hfq and somewhat different sets of prevalent RNA-RNA pairs. Our analysis provides insights into how the RNA-binding faces of Hfq contribute to pairing in vivo, documents the key role for the rim face in stabilizing RNA pairs on Hfq, and highlights intriguing differences captured by different RNA-RNA interactome approaches.