Small Regulatory RNAs

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

During the past 25 years, we 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. 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, many in collaboration with the group of Dr. Susan Gottesman, have allowed us to learn more about the mechanism by which the Hfq protein facilitates base pairing through multiple RNA binding domains. 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 (1). In addition to characterizing the proteins associated with base pairing sRNAs, we also have been studying the mechanisms by which base pairing alters gene expression. Most characterized interactions between bacterial small RNAs (sRNAs) and their target mRNAs occur near ribosome binding sites, resulting in changes in translation initiation or target mRNA decay. However, global RNA-RNA interactome approaches revealed that sRNA base pairing also occurs internal to coding sequences. In a recent study (2), we examined the impact of sRNA pairing to these internal sequences. Overexpression of the corresponding sRNA led to decreased target protein levels for two sRNA-mRNA pairs, but there were no differences for five others. By further examining ArcZ-ligA and ArcZ-hemK, we discovered that ArcZ pairing with the mRNAs leads to translation pausing and increased protein activity. A ligA point mutation that eliminates sRNA pairing resulted in increased sensitivity to DNA damage, revealing the physiological consequences of the regulation. Thus, regulatory RNA pairing in coding sequences can locally slow translation elongation, likely impacting co-translational protein folding and allowing improved incorporation of co-factors or more optimal folding under specific conditions. 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. Studies of these Hfq-binding sRNAs has given insights into the nuanced control of the regulatory networks as well as into bacterial physiology in general (3). 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. Similarly, we found 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. We also described four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor σ28 (fliA) and which have varied effects on flagellin protein levels, flagella number and cell motility. Intriguingly, MotR corresponding to the 5' UTR of an early gene in the flagella regulon, activates flagellar synthesis, while FliX, corresponding to a late gene in the flagella regulon, downregulates flagellar synthesis, illustrating how sRNA-mediated regulation can overlay a complex network enabling temporal control. 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 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). 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. 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. 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. 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.