Small RNAs (sRNAs) are major regulators of gene expression in bacteria, exerting their regulation in trans by base pairing with target RNAs. Traditionally, sRNAs were considered post-transcriptional regulators, mainly regulating translation by blocking or exposing the ribosome binding site. However, accumulating evidence suggest that sRNAs can exploit the base pairing to manipulate their targets in different ways, assisting or interfering with various molecular processes involving the target RNA. Currently there are a few examples of these alternative regulation modes, but their extent and implications in the cellular circuitry have not been assessed. Here we propose to take advantage of the power of RNA-seq-based technologies to develop innovative approaches to address these challenges transcriptome-wide. These approaches will enable us to map the regulatory mechanism a sRNA employs per target through its effect on a certain molecular process. For feasibility we propose studying three processes: RNA cleavage by RNase E, pre-mature Rho-dependent transcription termination, and transcription elongation pausing. Finding targets regulated by sRNA manipulation of the two latter processes would be especially intriguing, as it would suggest that sRNAs can function as gene-specific transcription regulators (alluded to by our preliminary results). As a basis of our research we will use the network of ~2400 sRNA-target pairs in Escherichia coli, deciphered by RIL-seq (a method we recently developed for global in vivo detection of sRNA targets). Revealing the regulatory mechanism(s) employed per target will shed light on the principles underlying the integration of distinct sRNA regulation modes in specific regulatory circuits and cellular contexts, with direct implications to synthetic biology and pathogenic bacteria. Our study may change the way sRNAs are perceived, from post-transcriptional to versatile regulators that apply different regulation modes to different targets.
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