Small Noncoding RNAs and Small ORFs

Gisela Storz, PhD

Gisela Storz, PhD, Head, Section on Environmental Gene Regulation
Aixia Zhang, PhD, Staff Scientist
Sylvain Durand, PhD, Postdoctoral Fellow
Fanette Fontaine, PhD, Postdoctoral Fellow
Elizabeth Fozo, PhD, Postdoctoral Fellow
Matthew Hemm, PhD, Postdoctoral Fellow
Errett Hobbs, PhD, Postdoctoral Fellow
Jason A. Opdyke, PhD, Postdoctoral Fellow
Lauren Waters, PhD, Postdoctoral Fellow
Jillian Astarita, BA, Predoctoral Fellow
Kathy S. Mendieta, BA, Predoctoral Fellow
Lisa M. Stamper, BS, Predoctoral Fellow

Currently, we have two main interests: (1) identification and characterization of small, noncoding RNAs and (2) identification and characterization of small ORFs. For several reasons, researchers have tended to overlook small RNAs and small proteins. Small, noncoding RNAs and small ORFs have been overlooked because they do not lend themselves to detection in biochemical assays. Most genome annotation misses the corresponding genes, which are poor targets for genetic approaches.

Identification of small, noncoding RNAs

Zhang, Storz; in collaboration with Gottesman

We have carried out several systematic screens for small, noncoding RNA genes in Escherichia coli. The screens are all applicable to other organisms. One approach—based on computer searches of intergenic regions for extended regions of conservation among closely related species—led to the identification of 17 conserved small RNAs. Another screen for small RNAs that coimmunoprecipitate with the RNA-binding protein Hfq allowed us to detect six less well-conserved RNAs. A third approach—size fractionation of total RNA followed by linker ligation and cDNA synthesis—resulted in the identification of still other small RNAs. We recently obtained tiled microarrays that provide coverage of the entire E. coli genome and are now using the arrays to extend our identification of small RNAs.

Storz G, Haas D. A guide to small RNAs in microorganisms. Curr Opin Microbiol 2007;10:93-95.

Characterization of specific small, noncoding RNAs

Durand, Fontaine, Fozo, Mendieta, Opdyke, Stamper, Waters, Zhang, Storz; in collaboration with Aravind, Kawano, Rudd

Increasingly, we have been focusing on elucidating the functions of the small RNAs in E. coli. We previously showed that the OxyS RNA, whose expression is induced in response to oxidative stress, represses translation by basepairing with target mRNAs. OxyS RNA action is dependent on the Smlike Hfq protein, which functions as a chaperone to facilitate OxyS RNA basepairing with its target mRNAs. We also discovered that the abundant 6S RNA binds to and modifies RNA polymerase. In addition, we elucidated the functions of the MicC RNA and GadY RNA, which also bind to Hfq and act by basepairing. We found that the MicC RNA represses translation of the OmpC outer membrane porin. Interestingly, under most conditions, the MicC RNA shows expression opposite that of the MicF RNA, which represses expression of the OmpF porin. Basepairing between the GadY RNA and the 3′-untranslated region (3′ UTR) of the gadX mRNA encoded opposite gadY leads to an increase in levels of the gadX mRNA and GadX protein. Increased GadX levels in turn result in increased expression of the acid-response genes controlled by the GadX transcription factor.

In one recent study, we characterized a small RNA (SymR). It is encoded in cis to an SOS-induced gene whose product shows homology to the antitoxin MazE (SymE). We demonstrated that synthesis of the SymE protein is tightly repressed at several levels by the LexA repressor, the SymR RNA, and the Lon protease. SymE co-purifies with ribosomes, and overproduction of the protein leads to cell growth inhibition, decreased protein synthesis, and increased RNA degradation. These properties are shared with several RNA endonuclease toxins of the toxin-antitoxin modules, and we reported that the SymE protein represents evolution of a toxin from AbrB fold proteins, which are typically antitoxins. We suggest that SymE promotion of RNA cleavage may be important for the recycling of RNAs damaged under SOS-inducing conditions.

In another recent study, we characterized the Sib RNAs, which are encoded by five repeats in E. coli K-12, though the number of repeats varies among E. coli strains. All five Sib RNAs in E. coli K-12 are expressed, and we observed no phenotype for a five-sib deletion strain. However, we observed a phenotype reminiscent of plasmid addiction for overexpression of the Sib RNAs. Further examination of the SIB repeat sequences revealed conserved open reading frames encoding highly hydrophobic 18–19 amino acid proteins (Ibs) opposite each sib gene. The Ibs proteins were toxic when overexpressed, although the toxicity could be prevented by co-expression of the corresponding Sib RNA. Two other RNAs encoded divergently in the yfhL-acpS intergenic region were similarly found to encode a small hydrophobic protein (ShoB) and an antisense RNA regulator (OhsC). Overexpression of both IbsC and ShoB led to immediate changes in membrane potential, suggesting that both proteins affect the cell envelope. Whole-genome expression analysis showed that overexpression of IbsC and ShoB, as well as of the small hydrophobic LdrD and TisB proteins, has both overlapping and unique consequences for the cell. We are currently engaged in studies characterizing the OxyS, GadY, and Sib RNAs and elucidating the roles of other newly discovered small RNAs.

Fozo EM, Kawano M, Fontaine F, Kaya Y, Mendieta KS, Jones KL, Ocampo A, Rudd KE, Storz G. Repression of small toxic protein synthesis by the Sib and OhsC small RNAs. Mol Microbiol 2008;70:1076-1093.
Kawano M, Aravind L, Storz G. An antisense RNA controls synthesis on an SOS-induced toxin evolved from an antitoxin. Mol Microbiol 2007;64:738-754.

Identification and characterization of small ORFs

Astarita, Fontaine, Hemm, Hobbs, Storz; in collaboration with Paul, Rudd, Schneider

In our genome-wide screens for small RNAs, we found that several short RNAs actually encode small proteins. The correct annotation of the smallest proteins is one of the greatest challenges of genome annotation. Perhaps more important, few annotated short ORFs have been confirmed to correspond to synthesized proteins. Although small proteins have been largely overlooked, the few small proteins subjected to detailed study in bacterial and mammalian cells have revealed important functions in signaling and in cellular defenses. Thus, we initiated a project to identify E. coli proteins of less than 50 amino acids and elucidate their functions by using many of the approaches with which our laboratory has characterized the functions of small, noncoding RNAs. We used sequence conservation and ribosome binding site models to predict genes encoding small proteins, which are defined as proteins with 16–50 amino acids, in the intergenic regions of the E. coli genome. We tested expression of the predicted and previously annotated small proteins by integrating the sequential peptide affinity tag directly upstream of the stop codon on the chromosome and then using immunoblot assays to assay for synthesis. Our approach confirmed the synthesis of 20 previously annotated and 18 newly discovered proteins of 16–50 amino acids. Remarkably, more than half of the newly discovered proteins are predicted to be single transmembrane proteins, of which nine co-fractionate with cell membranes. Systematic screens for (1) growth conditions that lead to increased expression and (2) phenotypes associated with null mutations are beginning to provide insights into the physiological roles of these small proteins.

Bender RA, Downs D, Kiley P, LaRossa RA, Sonenshein AL, Storz G. Bridges and chasms: summary of the IMAGE 2 meeting in Montreal, Canada, 30 April to 3 May 2007. J Bacteriol 2008;190:792-797.

Characterization of the OxyR and Fur transcription regulators

Storz; in collaboration with Schneider

Previously, a major focus of our laboratory was the characterization of the OxyR transcription regulator, particularly its sensitivity to oxidation and its binding to DNA. We concluded our work in this area with a study of OxyR mutants that defined a region where OxyR contacts RNA polymerase. In collaboration with Thomas Schneider, we also completed a computational analysis of DNA sites that bind to the iron repressor protein Fur.

Chen Z, Lewis KA, Shultzaberger RK, Lyakkhov IG, Zheng M, Doan B, Storz G, Schneider TD. Discovery of Fur binding site clusters in Escherichia coli by information theory models. Nucleic Acids Res 2007;35:6762-6777.

Collaborators

L. Aravind, PhD, Computational Biology Branch, National Center for Biotechnology Information, NLM, Bethesda, M
Susan Gottesman, PhD, Laboratory of Molecular Biology, NCI, Bethesda, MD
Mitsuoki Kawano, PhD, Omics Science Center, RIKEN Yokohama Institute, Kanagawa, Japan
Brian J. Paul, PhD, DuPont Central Research and Development, Wilmington, DE
Kenneth E. Rudd, PhD, University of Miami Miller School of Medicine, Miami, FL
Thomas D. Schneider, PhD, Center for Cancer Research Nanobiology Program, NCI, Frederick, MD

For further information, contact storz@helix.nih.gov or visit http://cbmp.nichd.nih.gov/segr.