Small Regulatory RNAs

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

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Abstract

During the past 20 years, we have carried out several different systematic screens for small regulatory RNAs in Escherichia coli. These screens have included computational searches for conservation of intergenic regions and direct detection after size selection or co-immunoprecipitation with RNA binding proteins. Most recently, we have been using deep sequencing approaches to map the 5' and 3' ends of all transcripts to further extend our identification of small RNAs in a range of bacteria species (1). This work has shown that sRNAs are encoded by diverse loci including sequences overlapping mRNAs. A major focus for the group has been to elucidate the functions of the small RNAs that we and others have identified. Early on we showed that the OxyS RNA, whose expression is induced in response to oxidative stress, acts to repress translation through limited base pairing with target mRNAs. We discovered OxyS action is dependent on the Sm-like Hfq protein, which acts as a chaperone to facilitate OxyS RNA base pairing with its target mRNAs. Follow up studies have allowed us to learn more about the mechanism by which the Hfq protein facilitates base pairing through multiple RNA binding domains (2). We also have started to explore the role of ProQ, a second RNA chaperone in E. coli and, by comparing the sRNA-mRNA interactomes by deep sequencing, found that ProQ and Hfq have overlapping as well as competing roles in the cell. It is likely that still other RNA binding proteins such as KH domain proteins are involved in small RNA-mediated regulation (3). Hfq-binding small RNAs, which act through limited base pairing, are integral to many different stress responses in E. coli and other bacteria as well as during the interaction between bacteria and bacteriophage. For example, we showed that the Spot 42 RNA, whose levels are highest when glucose is present, plays a broad role in catabolite repression by directly repressing genes involved in central and secondary metabolism, redox balancing, and the consumption of diverse non-preferred carbon sources. A recent collaborative study of Vibrio cholerae revealed that the QrrX RNA controls quorum sensing dynamics and biofilm formation (4). We also previously discovered that a Sigma(E)-dependent small RNA, MicL, transcribed from a promoter located within the coding sequence of the cutC gene represses synthesis of the lipoprotein Lpp, the most abundant protein in the cell, to oppose membrane stress. We documented that the copper sensitivity phenotype, previously ascribed to inactivation of the cutC gene, is actually derived from the loss of MicL and elevated Lpp levels. More recently, we have shown that a small RNA derived from the 3' UTR of the glnA encoding glutamine synthetase impacts E. coli growth under low nitrogen conditions by modulating the expression of genes that affect carbon and nitrogen flux (5). As more and more sRNAs encoded by 5' or 3' UTRs or internal to coding sequences are being found, our observations raise the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. One interesting recent observation is that some small RNAs have dual functions in that they act by both base pairing and encode a small, regulatory protein. For example, we discovered the Spot 42 RNA also encodes a 15-amino acid protein (denoted SpfP) (6). Overexpression of just the small protein from a Spot 42 derivative deficient in base-pairing activity, or just the base pairing activity from a Spot 42 derivative with a stop codon mutation both prevented growth on galactose, revealing that the small protein and the small RNA impact the same pathway. Copurification experiments showed that SpfP binds the CRP transcription factor, affecting the kinetics of induction when cells are shifted from glucose to galactose medium. Thus, the small protein reinforces the feedforward loop regulated by the base-pairing activity of the Spot 42 RNA. As a second example, we found a 164-nucleotide RNA previously shown to encode a 28-amino acid protein (denoted AzuC) also base pairs with the cadA and galE mRNAs to block expression (7). Interestingly, AzuC translation interferes with the observed repression of cadA and galE by the RNA, and base pairing interferes with AzuC translation, demonstrating that the translation and base-pairing functions compete. We hypothesize that many more dual-function RNAs remain to be discovered and suggest that they can be exploited to control gene expression at multiple levels. We successfully constructed a functional synthetic dual-function regulator from a small protein and a small protein encoded by adjacent genes and used this synthetic construct to study the functional organization of dual-function RNAs (8). In addition to small RNAs that act via limited base pairing, we have been interested in regulatory RNAs that act by other mechanisms. For instance, early work showed that the 6S RNA binds to and modulates RNA polymerase by mimicking the structure of an open promoter. In another study, we discovered that a broadly-conserved RNA structure motif, the yybP-ykoY motif, found in the 5-UTR of the mntP gene encoding a manganese exporter directly binds manganese, resulting in a conformation that liberates the ribosome-binding site. Further studies to characterize other Hfq- and ProQ-binding RNAs and their physiological roles and evolution as well as regulatory RNAs that act in ways other than base pairing are ongoing.