# Riboregulation in Nitrogen-Fixing Endosymbiotic Bacteria
## 2 Deciphering the Rhizbial Non-Coding Transcriptome: From Comparative Genomics to RNA-Seq  
Non-protein coding genes largely escape classical genetics screens and the primary annotation of a single bacterial genome sequence, which is essentially limited to the prediction of open reading frames (ORFs), tRNAs, and rRNAs. Before the advent of high-throughput sequencing, computational comparative genomics was thus the tool of choice to identify conserved regions with putative functions in the unannotated portions of the genome. Accordingly, pioneering seminal genome-wide searches for sRNAs in rhizobia relied on the comparison of intergenic sequences (i.e., genomic regions between ORFs; IGRs) from phylogenetically close species to unveil sRNAs of the _trans_-acting class. Specifically, three studies published almost concurrently used the IGRs from the _S. melioti_ reference strain 1021 (Rm1021) as queries to interrogate the genomes of \(\alpha\)-proteobacterial relatives, i.e., plant symbionts (e.g., _Mesorhizobium loti_, _Rhizobium etli_ or _R. leguminosarum_ bv. _viciae_), phytopathogens (_Agrobacteriumum tumefaciens_) and animal pathogens (_Brucella_ species) [18; 19; 20]. All three searches combined genomic comparisons with other known features of _trans_-sRNAs, namely association with orphan transcription signatures (promoter motifs and/or Rho-independent transcriptional terminators) and conservation of thermodynamically stable secondary structures. Collectively, these approaches predicted more than a hundred IGRs in the three replicons of the Rm1021 genome (chromosome, and pSymA and pSymB megaplasmids) putatively encoding a _trans_-sRNA. Northern blot hybridization of total RNA, RACE (Rapid Amplification of cDNA Ends) mapping of transcripts boundaries and/or microarray probing experimentally confirmed that a few dozens of these candidate IGRs did express sRNA species from independent transcription units. These studies also anticipated the symbiotic and/or stress-dependent expression of subsets of the identified sRNAs. In the absence of further functional insights, these _trans_-sRNAs were initially referred to as either Smr [20], Sra [18] or Sm [19] (Table 1). A similar combination of in silico searches and experimental approaches also delivered the first inventories of sRNAs expressed by _R. etli_ CFN42 (termed ReC sRNAs), _Bradyrhizobium japonicum_ USDA110 (BirC sRNAs) and _M. huakui_ 7653R (MH_s sRNAs) [21; 22; 23]. These rhizobia are the symbiotic partners of common bean (_Phaseolus vulgaris_), soybean (_Glycine max_), and milkvetch (_Astragalus sinicus_), respectively. Inherent limitations to both computational searches and microarray designs necessarily biased these early genome-wide screens for sRNAs in rhizobia to the identification of putative intergenic, _trans_-acting, conserved riboregulators. Straightforward experimental identification of TSS associated to coding sequences, untranslated mRNA regions, and non-coding RNA genes in the prokaryotic genomes is now feasible with the implementation of oriented differential RNA-Seq (dRNA-Seq) [24] or Cappable-Seq [25] strategies. In particular, dRNA-Seq surveys rediscovered the early-identified sRNAs in _S. melloti_ and _B. japonicum_ and further uncovered the complex rhizobial transcriptomes by adding hundreds of unknown _trans_-sRNAs, as well as thousands of mRNA-derived sRNAs and sRNAs [26; 27; 28; 29]. Other RNA-Seq studies are conceived to profile specific subpopulations of cellular transcripts supposedly enriched in sRNAs, e.g., RNA species co-immunoprecipitation with the major bacterial RNA chaperone Hfq [30; 31]. However, this approach resulted in a minor addition to the sRNA landscape revealed by deep-sequencing of _S. melloti_ total RNA [32]. Prokaryotic gene prediction pipelines such as EuGen-P have incorporated the novel gene structural features uncovered by dRNA-Seq for the accurate reannotation of the _S. melloti_ (strains Rm1021 and Rm2011) and _B. japonicum_ USDA110 genomes [27; 28; 29].