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Post-transcriptional control provides bacterial pathogens a method by which they can rapidly adapt to environmental change. Dual exo- and endonucleolytic activities of RNase J enzymes contribute to Gram-positive RNA processing and decay. First discovered in Bacillus subtilis, RNase J1 plays a key role in mRNA maturation and degradation, while the function of the paralogue RNase J2 is largely unknown. Previously, we discovered that deletion of the Enterococcus faecalis rnjB gene significantly attenuates expression of a major virulence factor involved in enterococcal pathogenesis, the Ebp pili. In this work, we demonstrate that E. faecalis rnjB encodes an active RNase J2, and that the ribonuclease activity of RNase J2 is required for regulation of Ebp pili. To further investigate how rnjB affects E. faecalis gene expression on a global scale, we compared transcriptomes of the E. faecalis strain OG1RF with its isogenic rnjB deletion mutant (ΔrnjB). In addition to Ebp pili regulation, previously demonstrated to have a profound effect on the ability of E. faecalis to form biofilm or establish infection, we identified that rnjB regulates the expression of several other genes involved in bacterial virulence and fitness, including gls24 (a virulence factor important in stress response). We further demonstrated that the E. faecalis RNase J2 deletion mutant is more sensitive to bile salt and greatly attenuated in in vivo organ infection as determined by an IV-sublethal challenge infection mouse model, indicating that E. faecalis RNase J2 plays an important role in E. faecalis virulence.

In eukaryotes, the exosome plays a central role in RNA maturation, turnover, and quality control. In Saccharomyces cerevisiae, the core exosome is composed of nine catalytically inactive subunits constituting a ring structure and the active nuclease Rrp44, also known as Dis3. Rrp44 is a member of the ribonuclease II superfamily of exoribonucleases which include RNase R, Dis3L1 and Dis3L2. In this work we have functionally characterized three residues located in the highly conserved RNB catalytic domain of Rrp44: Y595, Q892 and G895. To address their precise role in Rrp44 activity, we have constructed Rrp44 mutants and compared their activity to the wild-type Rrp44. When we mutated residue Q892 and tested its activity in vitro , the enzyme became slightly more active. We also showed that when we mutated Y595, the final degradation product of Rrp44 changed from 4 to 5 nucleotides. This result confirms that this residue is responsible for the stacking of the RNA substrate in the catalytic cavity, as was predicted from the structure of Rrp44. Furthermore, we also show that a strain with a mutation in this residue has a growth defect and affects RNA processing and degradation. These results lead us to hypothesize that this residue has an important biological role. Molecular dynamics modeling of these Rrp44 mutants and the wild-type enzyme showed changes that extended beyond the mutated residues and helped to explain these results. © 2013 Reis et al., FCT, Portugal (PEst-OE/EQB/LA0004/2011); European Commission (FP7-KBBE-2011-1-289326, FP7 HEALTH-F3-2009-223431, FP7 HEALTH-2011-278603); National Institutes of Health (R01GM099790); Welch foundation (AU-177); the Spanish Ministerio de Ciencia e Innovación (SAF2007-61926, IPT2011-0964-900000, SAF2011-13156-E); Fundación Ramón Areces; Centro de Computación Científica CCC-UAM; European Social Fund