# Riboregulation in Nitrogen-Fixing Endosymbiotic Bacteria
## 6 Proteins Assisting sRNA Activity
### Rhizbial Ribonucleases  
As major determinants of the steady-state level of all cellular transcripts, ribonucleases (RNases) are critical elements in post-transcriptional regulatory networks [126]. Decay of mRNA upon antisense interaction with regulatory RNAs commonly involves the prevalent prokaryotic endoribonucleases RNase E and/or RNase III, which are specific to single- and double-stranded RNAs (ssRNA and dsRNA), respectively [127]. In some cases, RNase E is recruited by Hfq to the interplay between a _trans_-sRNA and its target(s) as a component of a multiprotein complex called RNA degradosome that also contains a 3\({}^{\prime}\)-exoribonuclease (polynucleotide phosphorylase or PNPase), a RNA helicase (RhIB) and an enolase as the core proteins [126]. On the other hand, RNase III preferentially recognizes RNA duplexes, thus being a major player of gene silencing promoted by asRNAs [128]. RNA decay initiated by RNase E or RNase III results into intermediate products that are further degraded by other cellular endo- and exoribonucleases, e.g., PNPase, RNase II or RNase R [127].  
Rhizobial genomes encode a set of more than 20 ribonucleases that include all the aforementioned proteins, but for the vast majority there is a lack of functional information [129]. Nonetheless, several reports have anticipated the involvement of RNase E in _trans_-sRNA stability and regulation in both Hfq-dependent and independent manner [81; 82; 87; 130; 131]. RNase III and RNase J, which is not present in _E. coli_, are both involved in rRNA maturation but their putative role in riboregulation has not been investigated yet [132; 133]. RNase III encoded by the _S. meliloti_ _mc_ gene has been biochemically and genetically characterized [134]. Its catalytic features resemble those of the _E. coli_ ortholog but with different requirements for optimal activity regarding metal co-factor preference or pH tolerance that could be related to the rhizobial lifestyle. Indeed, although _mc_ deletion does not compromise viability and morphology of _S. meliloti_ cells, it is detrimental for growth and overall symbiotic performance on alfalfa roots [134].  
The gene annotated as _SMc01113_ in the chromosome of the _S. meliloti_ reference strain Rm1021 is almost ubiquitous in bacteria and has been included in the proposed minimal prokaryotic genome [135]. It encodes a putative metal-dependent hydrolase, later called YbeY, that shares predicted structural similarities with the MID domain of the Argonaute (AGO) proteins involved in eukaryotic RNA silencing, but its function has remained elusive until recently [136]. _S. meliloti_ Hfq and YbeY mutants show strikingly similar free-living and symbiotic phenotypes as well as misregulation of several predicted sRNA-mRNA regulatory pairs [136; 137]. These structural and phenotypic features initially hinted at an Hfq-like role of YbEY in riboregulation [136]. However, the _E. coli_ YbeV ortholog was later shown to be a metal-dependent endoribonuclease that specifically acts on ssRNA [138]. Biochemical characterization of _S. meliloti_ YbeV also supports a universal role of this protein as RNase [139; 140]. However, unlike its enterobacterial ortholog, _S. meliloti_ YbeV is also competent for cleaving dsRNA [139]. Transcriptome alterations in the _S. meliloti_ YbeV mutant predict a role of this enzyme in _trans_-sRNA and asRNA mediated silencing of genes involved in nutrient uptake and symbiotic N fixation, respectively [139]. In this regard, both in vivo and in vitro evidences suggest that YbeV is required for AbcR2-dependent down-regulation of the ABC amino acid transporter _prbA_ mRNA. Further supporting a role in dsRNA degradation, _S. meliloti_ YbeV has been also shown to co-purify with several asRNAs and their mRNA partners, including the symbiotically-relevant _nifA_[139]. It would be interesting to test whether the alteration of asRNA-mediated regulation constitutes one of the main drivers of the symbiotic phenotype associated to YbeV activity.