Fitness and Functional Landscapes of RNase III

The RNase III family of enzymes consists of highly conserved endoribonucleases that cleave dsRNA in organisms as simple as bacteria to as complex as humans. Within the E. coli cell, RNase III is a globally regulating enzyme that differentially regulates a myriad of genes through cleavage of dsRNA, including its own regulation through cleavage of a stem loop in its own 5’ UTR. We harnessed this regulatory power by fusing the 5’-UTR of RNase III and gGFP, a guide RNA specific for the GFP gene, to create a dCas9-based in vivo selection for RNase III activity where full GFP repression is only achieved when the 5’UTR is cleaved from the gGFP. We used the selection to study, through deep mutational scanning, the effects of all single-codon mutations to RNase III, generating a landscape of functional scores. Through growth competition in liquid media, and solid media after transformation, we measured the fitness effects and transformation scores of all single-codons mutations, as well. Our study represents the first that reports on the comprehensive fitness effects of a globally regulating enzyme in its native organism and the first to comparatively analyze the fitness and functional scores of nearly all mutations in the same gene. We found that the distribution of fitness effects for RNase III was bimodal, with peaks centered around mutations with deleterious and neutral effects, like other comprehensively studied proteins. We also found that fitness and functional scores were positively correlated, meaning that, in general, fitness is dependent on the ability for RNase III to cleave dsRNA properly. Our study found that the RNase III’s RNase III domain (RIIID), which contains the RNase III signature motif and all active site residues, was much more sensitive to mutation than its dsRNA binding domain (dsRBD), which is responsible for recognition and binding to dsRNA. We also found that fitness was buffered to small effects on RNase III activity. Differential effects on fitness and functional scores for mutations at F188 suggest that it may be important for RNase III cleavage specificity. Despite being one of the simplest enzymes in the RNase III family, homology between RNase III-like enzymes indicates that our approach and results might be applied to enzymes from more complex organisms, such as Dicer or Drosha