Superfolder GFP reporters validate diverse new mRNA targets of the classic porin regulator, MicF RNA

MicF is a textbook example of a small regulatory RNA (sRNA) that acts on a trans-encoded target mRNA through imperfect base pairing. Discovery of MicF as a post-transcriptional repressor of the major Escherichia coli porin OmpF established the paradigm for a meanwhile common mechanism of translational inhibition, through antisense sequestration of a ribosome binding site. However, whether MicF regulates additional genes has remained unknown for almost three decades. Here, we have harnessed the new superfolder variant of GFP for reportergene fusions to validate newly predicted targets of MicF in Salmonella. We show that the conserved 5' end of MicF acts by seed pairing to repress the mRNAs of global transcriptional regulator Lrp, and periplasmic protein YahO, while a second targeting region is also required to regulate the mRNA of the lipid A-modifying enzyme LpxR. Interestingly, MicF targets lpxR at both the ribosome binding site and deep within the coding sequence. MicF binding in the coding sequence of lpxR decreases mRNA stability through exacerbating the use of a native RNase E site proximal to the short MicF-lpxR duplex. Altogether, this study assigns the classic MicF sRNA to the growing class of Hfq-associated regulators that use diverse mechanisms to impact multiple loci.