SMALL, NONCODING RNAS AND SMALL ORFS
, Head, Section on Environmental Gene Regulation
Aixia Zhang, PhD, Staff Scientist
Elizabeth Fozo, PhD, Postdoctoral Fellow
Matthew Hemm, PhD, Postdoctoral Fellow
Errett Hobbs, PhD, Postdoctoral Fellow
Mitsuoki Kawano, PhD, Postdoctoral Fellow
Jason A. Opdyke, PhD, Postdoctoral Fellow
Brian Paul, PhD, Postdoctoral Fellow
Lauren Waters, PhD, Postdoctoral Fellow
Lisa M. Stamper, BS, Predoctoral Fellow
Emily Yen, BA, Predoctoral Fellow
Identification of small, noncoding RNAs
We have been carrying out several different systematic screens for small, noncoding RNA genes in E. 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-conserved RNAs. A third approach—size fraction of total RNA followed by linker ligation and cDNA synthesis—resulted in the identification of still other small RNAs. We have recently obtained tiled microarrays that provide coverage of the whole E. coli genome, and we are using the microarrays 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.
Development of general approaches for the characterization of small, noncoding RNAs
We also have been developing tools for the characterization of small RNA regulators. Detection of RNAs on microarrays has become a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. In collaboration with Susan Gottesman and Stephen Leppla, we developed an approach that bypasses the limitations of current methods. In our approach, unmodified RNA is hybridized directly to DNA microarrays for detection with the high-affinity, nucleotide sequence–independent, DNA/RNA hybrid–specific mouse monoclonal antibody. Subsequent reactions with a fluorescently labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG, together with fluorescently labeled streptavidin, produce a signal that can be measured in a standard microarray scanner. The antibody-based method detected low-abundance small RNAs of E. coli much more efficiently than the commonly used cDNA-based method.
Many bacterial sRNAs act as post-transcriptional regulators by base pairing with target mRNAs. While the number of characterized small RNAs has steadily increased, only a limited number of corresponding mRNA targets have been identified. In collaboration with Susan Gottesman and Brian Tjaden, we developed and tested a program, TargetRNA, that predicts the targets of these small RNA regulators. We evaluated the program by assessing whether previously known targets could be identified. We then used the program to predict targets for the E. coli RNAs RyhB, OmrA, OmrB, and OxyS and compared the predictions with changes in whole genome expression patterns observed upon expression of the small RNAs.
Hu Z, Zhang A, Storz G, Gottesman S, Leppla SH. An antibody-based microarray assay for small RNA detection. Nucleic Acids Res 2006;34:e52.
Tjaden B, Goodwin SS, Opdyke JA, Guillier M, Fu DX, Gottesman S, Storz G. Target prediction for small, noncoding RNAs in bacteria. Nucleic Acids Res 2006;34:2791-802.
Characterization of specific small, noncoding RNAs
We have grown increasingly interested in elucidating the functions of the small RNAs in E. coli. We previously showed that OxyS RNA, whose expression is induced by OxyR in response to oxidative stress, acts to repress translation by base pairing with target mRNAs. OxyS RNA action is dependent on the Sm-like Hfq protein, which functions as a chaperone to facilitate OxyS RNA base pairing 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 two other small RNAs, the MicC RNA and the GadY, that also bind to Hfq and act by base pairing. We found 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. Base pairing between the GadY RNA and the 3′-untranslated region (3′ UTR) of the gadX mRNA encoded opposite gadY leads to increased 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.
Recently, we characterized a small RNA (SymR) that is encoded in cis to an SOS-induced gene whose product shows homology to the antitoxin MazE (SymE). We showed 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 the AbrB fold, whose representatives are typically antitoxins. We suggest that SymE promotion of RNA cleavage may be important for the recycling of RNAs damaged under SOS-inducing conditions. Studies to characterize further the OxyS, GadY, and SymR RNAs and to elucidate the roles of other newly discovered small RNAs are ongoing.
Guillier M, Gottesman S, Storz G. Modulating the outer membrane with small RNAs. Genes Dev 2006;20:2338-48.
Kawano M, Aravind L, Storz G. An antisense RNA controls synthesis on an SOS-induced toxin evolved from an antitoxin. Mol Microbiol 2007;64:738-54.
Storz G, Gottesman S. Versatile roles of small RNA regulators in bacteria. In: Gesteland RF, Cech TR, Atkins JF, eds. The RNA World, Third edition. Cold Spring Harbor Press, 2006;567-94.
Storz G, Opdyke JA, Wassarman KM. Regulating bacterial transcription with small RNAs. Cold Spring Harb Symp Quant Biol 2007;71:269-73.
Characterization of small ORFs
In our genome-wide screens for small RNAs, we found that several short RNAs encode small proteins. Although small proteins have largely been overlooked, the few small proteins that have been studied in detail in bacterial and mammalian cells have been shown to play important functions in signaling and 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 the group has used to characterize the functions of small, noncoding RNAs.
Characterization of the OxyR and Fur transcription regulators
Previously, a major focus of the laboratory was the characterization of the OxyR transcription regulator, its sensitivity to oxidation, and its binding to DNA. In the past year, we also completed a long-standing study of OyxR mutants to define a region where OxyR contacts RNA polymerase. In collaboration with Thomas Schneider, we also completed a computational analysis of DNA binding sites for 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;35:6762-77.
Wang X, Mukhopadhyay P, Wood MJ, Outten FW, Opdyke JA, Storz G. Mutational analysis to define an activating region on the redox-sensitive transcriptional regulator OxyR. J Bacteriol 2006;188:8335-42.
COLLABORATORS
Shoshy Altuvia, PhD, The Hebrew University-Hadassah Medical School, Jerusalem, Israel
L. Aravind, PhD, Computational Biology Branch, National Center for Biotechnology Information, NLM, Bethesda, MD
Susan Gottesman, PhD, Laboratory of Molecular Biology, NCI, Bethesda, MD
Stephen H. Leppla, PhD, Laboratory of Bacterial Diseases, NIAID, Bethesda, MD
Thomas D. Schneider, PhD, Center for Cancer Research Nanobiology Program, NCI, Frederick, MD
Brian Tjaden, PhD, Wellesley College, Wellesley, MA
Karen M. Wassarman, PhD, University of Wisconsin, Madison, WI
For further information, contact storz@helix.nih.gov.
