Transcriptome-Wide Analysis of Stationary Phase Small ncRNAs in E. coli

Abstract

Almost two-thirds of the microbiome’s biomass has been predicted to be in a non-proliferating, and thus dormant, growth state. It is assumed that dormancy goes hand in hand with global downregulation of gene expression. However, it remains largely unknown how bacteria manage to establish this resting phenotype at the molecular level. Recently small non-protein-coding RNAs (sRNAs or ncRNAs) have been suggested to be involved in establishing the non-proliferating state in bacteria. Here, we have deep sequenced the small transcriptome of Escherichia coli in the exponential and stationary phases and analyzed the resulting reads by a novel biocomputational pipeline STARPA (Stable RNA Processing Product Analyzer). Our analysis reveals over 12,000 small transcripts enriched during both growth stages. Differential expression analysis reveals distinct sRNAs enriched in the stationary phase that originate from various genomic regions, including transfer RNA (tRNA) fragments. Furthermore, expression profiling by Northern blot and RT-qPCR analyses confirms the growth phase-dependent expression of several enriched sRNAs. Our study adds to the existing repertoire of bacterial sRNAs and suggests a role for some of these small molecules in establishing and maintaining stationary phase as well as the bacterial stress response. Functional characterization of these detected sRNAs has the potential of unraveling novel regulatory networks central for stationary phase biology.

Transfer RNA-Derived Fragments, the Underappreciated Regulatory Small RNAs in Microbial Pathogenesis

“…Co-culture of human 5' tRNA halves with F. nucleatum inhibits bacterial growth, likely through interference with bacterial protein biosynthesis (He et al, 2018). Several studies have also demonstrated that environmental stress increases cytosolic tRNA halves in microbes (Thompson et al, 2008;Garcia-Silva et al, 2010;Fricker et al, 2019;Raad et al, 2021); however, the function of 5' and 3' tRNA halves induced by stress remains elusive.…”

“…ContinuedRaad et al, 2021 of note. Is the production and packaging of tRNA fragments into microbial EVs regulated?…”

The stationary phase-specific sRNAfimR2is a multifunctional regulator of bacterial motility, biofilm formation and virulence

“…The CsrA-interacting sRNA CsrB (Liu et al, 1997) was also recovered in the same fractions (Figure 5A). However, full-length tRNA Gly and its 5'-tRNA fragment, a stationary-phase enriched sRNA that we previously recovered (Raad et al, 2021), were both largely depleted from these fractions (Figure 5A) thus highlighting the specificity of the experimental system. Taken together, these pulldown data confirmed that fimR2 and CsrA interact in vivo.…”

“…This suggested that the sRNA is important for stationary phase-specific phenomena. To gain insight into possible fimR2 functions, we ectopically expressed this sRNA in an inducible manner during exponential phase (Raad et al, 2021).…”

Ribosome-Associated ncRNAs (rancRNAs) Adjust Translation and Shape Proteomes

“…The initial fortuitous finding of ribosome-bound ncRNAs in polysome-derived RNA-sequencing data [ 33 ], which were probably prematurely referred to as ‘contaminants’, has fostered more dedicated research on the ribosomal ncRNA interactome. In the recent past, our laboratory conducted several targeted RNome screens for ribosome-associated ncRNA in various model organisms spanning all three domains of life ( Figure 2 ) [ 15 , 20 , 21 , 34 , 35 ]. To detect novel ncRNAs putatively regulating the ribosome’s activity, various stress conditions were applied to the different model systems.…”