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 RNA genes in Escherichia coli, which have shown that sRNAs are encoded by diverse loci including sequences overlapping mRNAs (1). These screens have included computational searches for conservation of intergenic regions and direct detection after size selection or co-immunoprecipitation with the RNA binding protein Hfq. 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 (2). 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. We have also 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 clear that 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 (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 nonpreferred carbon sources. Similarly, we 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 found 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. As more and more sRNAs encoded by 5' or 3' UTRs or internal to coding sequences are being found, our observations regarding MicL raise the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. 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.