# Transcriptome-Wide Analysis of Stationary Phase Small ncRNAs in _E. coli_
## 3 Discussion
### tRF Enrichment in Stationary Phase  
Interestingly, several tRNA-derived RNA fragments (tRFs) were enriched in the stationary phase (Figures 3a and 4a). Recently, tRFs have gained vast recognition as regulatory molecules in all three domains of life [51]. tRFs appear to be involved in a wide range of biological roles, including the regulation of transcription, translation, stress granule formation, apoptosis, cell proliferation, tumor suppression, RNAi, vesicle-mediated intercellular communication, intergenerational inheritance, retrotrans mobility, cell differentiation, and ribosome biogenesis [52; 53; 54; 55]. What kind of functions can these RNA molecules serve in simpler forms of life, such as bacteria? Bacterial tRFs, while not yet characterized to an equally detailed level compared to other domains, appear to be involved in a more complex level of gene expression regulation. A 3\({}^{\prime}\)-ETS (external transcribed spacer) of a pre-tRNA in _E. coli_ associates with the RNA chaperone Hfq, however, not to regulate gene expression as most Hfq-associated sRNAs do, but to sequester sRNAs away from their mRNA targets, thereby preventing transcriptional noise [56]. The same study suggests several other potential tRFs that are likely functional based on the conservation of tRNA ETS and ITS (internal transcribed spacer) sequences among enterobacteria. Our validated sequencing data for several tRFs in _E. coli_ confirmed the stationary phase-specific enrichment of these processing products. However, the functional scope of these tRF candidates likely surpassed that of serving as decoys as suggested earlier. Indeed, in the archeon _Haloferax volcanati_, a 5\({}^{\prime}\)-tRF was produced under alkaline stress conditions and downregulates gene expression on a global level by binding the small ribosomal subunit, thus interfering with mRNA loading [36]. Most of these tRFs were enriched in total RNA libraries, and expression profiling shows that these molecules were likely not binding the ribosome directly in _E. coli_ (Figures 3b and 4b). Unlike its peers, \(tRF^{AluV}\) was enriched in ribosome-associated RNA libraries (Supplementary Table S3), but strikingly, Northern blot analyses showed that this fragment was not interacting with ribosomes. One explanation for this discrepancy between sequencing data and experiments may be that \(tRF^{AluV}\) was interacting with other RNA molecules or protein partners closely associating with the ribosome, and as such, was temporally captured in our ribosome-associated sRNA libraries. In fact, the majority of the differentially expressed tRFs in our analysis may be interacting with RNA partners or RNA binding proteins; for instance, \(tRF^{Gly}\) (Figure 4a) was enriched in ProQ RIL-seq (RNA interaction by ligation and sequencing) and deletion experiments [57]. This suggests that this tRF is interacting with the chaperone ProQ and other partners, likely RNA molecules. Overall, the stationary phase-specific expression behavior of these tRNA fragments argues for putative physiological roles, and further functional analysis needs to be conducted to understand if these molecules are serving as molecular decoys or trans-encoded sRNAs.  
In this study, we surveyed the small transcriptomes of _E. coli_ in both the exponential and stationary phases by deep sequencing and showed that the composition of these sRNAs differed between both growth stages. Our refined biocomputational approach STARPA allowed the straightforward identification of several stable sRNAs that were likely aiding in the establishment of the resting phenotype or even orchestrating its establishment as part of regulatory networks. We suggest several sRNAs for further functional characterization, including abundant uncharacterized tRF species whose roles remain enigmatic in the bacterial kingdom. To uncover the function of these tRFs (and other sRNA candidates identified in our stationary phase sRNome study), a combination of molecular, biochemical, and genetic approaches is required.