Identification of YbeY-Protein Interactions Involved in 16S rRNA Maturation and Stress Regulation in Escherichia coli

Abstract

YbeY is part of a core set of RNases in Escherichia coli and other bacteria. This highly conserved endoribonuclease has been implicated in several important processes such as 16S rRNA 3′ end maturation, 70S ribosome quality control, and regulation of mRNAs and small noncoding RNAs, thereby affecting cellular viability, stress tolerance, and pathogenic and symbiotic behavior of bacteria. Thus, YbeY likely interacts with numerous protein or RNA partners that are involved in various aspects of cellular physiology. Using a bacterial two-hybrid system, we identified several proteins that interact with YbeY, including ribosomal protein S11, the ribosome-associated GTPases Era and Der, YbeZ, and SpoT. In particular, the interaction of YbeY with S11 and Era provides insight into YbeY’s involvement in the 16S rRNA maturation process. The three-way association between YbeY, S11, and Era suggests that YbeY is recruited to the ribosome where it could cleave the 17S rRNA precursor endonucleolytically at or near the 3′ end maturation site. Analysis of YbeY missense mutants shows that a highly conserved beta-sheet in YbeY—and not amino acids known to be important for YbeY’s RNase activity—functions as the interface between YbeY and S11. This protein-interacting interface of YbeY is needed for correct rRNA maturation and stress regulation, as missense mutants show significant phenotypic defects. Additionally, structure-based in silico prediction of putative interactions between YbeY and the Era-30S complex through protein docking agrees well with the in vivo results.

C21orf57 is a human homologue of bacterial YbeY proteins

“…Our findings suggest that the toxicity of HuYbeY expression in yeast is most likely not linked to its RNase activity, but rather to unfavorable or non-regulated interactions of HuYbeY with proteins or nucleic acids, ultimately affecting cell physiology. These observations are consistent with the notion that HuYbeY function in human cells must be directed and regulated by evolved partners as YbeY function is in bacteria [25] and are a first indication for the possible involvement of HuYbeY in human diseases that are associated with the dysregulation of cellular HuYbeY levels.…”

“…Second, we recently showed that certain residues (e.g. D85) located in a 4-stranded beta-sheet outside of the active site are important for interactions between EcYbeY and ribosomal protein S11; these interactions with the ribosome were also found to be important for EcYbeY’s role in rRNA maturation and stress regulation [25]. In addition, bacterial YbeY proteins share structural similarities with the MID domain of Argonaute (AGO) proteins, consistent with the observed involvement of YbeY in both Hfq-dependent and Hfq-independent small-RNA regulation in bacteria [17,20,28].…”

“…Interestingly, reduction of YbeY expression levels in V. cholerae results in defects in rRNA processing and ribosome quality control, changes in small RNA regulation, decreased stress tolerance, and ultimately a strongly compromised virulence potential [17]. Most recently, we have used bacterial 2-hybrid analysis to show that EcYbeY interacts with multiple partners including ribosomal protein S11, the GTPases Era and Der, the stress regulator SpoT and its operon partner YbeZ [25]. Interactions with S11 and Era appear to target the relatively non-specific endoribonuclease activity of YbeY during processing of the 3′-terminus of 16S rRNA, suggesting that interactions with various partner proteins are likely to be important for YbeY’s other physiological functions as well [25].…”

“…Most recently, we have used bacterial 2-hybrid analysis to show that EcYbeY interacts with multiple partners including ribosomal protein S11, the GTPases Era and Der, the stress regulator SpoT and its operon partner YbeZ [25]. Interactions with S11 and Era appear to target the relatively non-specific endoribonuclease activity of YbeY during processing of the 3′-terminus of 16S rRNA, suggesting that interactions with various partner proteins are likely to be important for YbeY’s other physiological functions as well [25]. …”

Identification of flavonoids as regulators of YbeY activity in Liberibacter asiaticus

“…YbeY also interacts with ribosomal proteins Era and S11 in E . coli , providing further evidence for its involvement in rRNA processing (Vercruysse et al ., ; Ghosal et al ., ).…”