# Transcriptome-Wide Analysis of Stationary Phase Small ncRNAs in _E. coli_
## 3 Discussion
### sRNAs in Stationary Phase Biology
However, several of these processing products enriched in ribosome-associated sRNA libraries did not derive from abundant rRNA transcripts (Supplementary Figure S3). This suggests that these molecules were protected from degradation by binding the ribosome [32] and may thus act as ranck RNAs. While ranck RNAs are poorly characterized in bacteria, very recently, an antitoxin RNA has been identified as ranck RNA in _Staphylococcus aureus_ that downregulates global protein synthesis by affecting tRNA binding, which, in turn, promotes persister cell formation [43]. As such, our enriched ribosome-associated sRNAs could play similar roles in dimming translation, a critical process for establishing and maintaining bacterial stationary phase. Our analysis also reported previously characterized sRNAs with important roles in the stationary phase, particularly enriched in total RNA libraries. For instance, and not so surprisingly, our differential sRNA analysis positioned _65 RNA_ as one of the most enriched sRNAs in the stationary phase (Supplementary Table S3). _65 RNA_ directly associates with RNA polymerase and its sigma factor 70, \(\sigma^{70}\), following its accumulation in the stationary phase and guides the downregulation of several \(\sigma^{70}\)-dependent promoters [44], a process coupled to the upregulation of RpoS-dependent transcripts. Furthermore, 65 RNA regulates the expression of several genes involved in metabolism [45], positioning this sRNA as a key regulator in establishing and maintaining bacterial dormancy. _sibC_ is a cis-acting sRNA encoded antisense to its target mRNA _ibSc_ coding for a toxin protein that is primarily involved in growth arrest [46]. The downregulation of the _ibSc_-encoded toxin expression in the stationary phase is coupled to the upregulation of the sRNA [47]. This growth-phase dependent expression of _sibC_ remains poorly understood. _ChiX_ is a transacting sRNA that strictly relies on Hfq to downregulate the expression of its targets in the stationary phase [48,49]. As our analysis captured several sRNAs with diverse regulatory mechanisms, we also predicted that the so-far uncharacterized portion of this sRNome could potentially be functional in the stationary phase. Out of the sRNA candidates that we validated experimentally, we also noted that the abundant _sRNA_35_ (Figure 3a) appeared to play a role in the stationary phase. Its overexpression is coupled to the upregulation of _rpoS_ (Figure 6c), a process innate to stationary phase [8] when it regulates transcription of about 500 stationary phase genes [50]. RpoS is not only known as a master regulator of general stress response in bacteria but its upregulation has also been connected to elevated persister cell formation [9; 10]. While it is not clear if our observed up-regulation of _rpoS_ was direct or indirect, _sRNA_35_ appeared to be a functional stationary phase-specific sRNA with a role yet to be unraveled. More dedicated experimental work is required to gain functional insight into the roles of this and other sRNAs identified in this study.