Gisela Storz, PhD, Head, Section on Environmental Gene Regulation
Aixia Zhang, PhD, Staff Scientist
Elizabeth Fozo, PhD, Postdoctoral Fellow
Matthew Hemm, PhD, Postdoctoral Fellow
Mitsuoki Kawano, PhD, Postdoctoral Fellow
Jason A. Opdyke, PhD, Postdoctoral Fellow
Brian Paul, PhD, Postdoctoral Fellow
Juan Miranda Rios, PhD, Guest Researcher
Nima Soltanzad, AB, Predoctoral Fellow
Emily Yen, BA, Predoctoral Fellow

We currently have two main interests: the identification and characterization of small, noncoding RNAs and the identification and characterization of small ORFs. Both classes of genes are frequently overlooked because they are missed by most genome annotation, they are poor targets for genetic approaches, and they are not detected in biochemical assays.

Identification of small, noncoding RNAs

Kawano, Miranda Rios, Storz; in collaboration with Martin, Rosner

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, which is 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 co-immunoprecipitate with the RNA-binding protein Hfq allowed us to detect six less well-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. In collaboration with Judah Rosner and Robert Martin, we also carried out an expression-based screen to detect small RNAs encoded within or on the opposite strand of protein-coding genes. We fused chromosomal fragments of approximately 160 bp to a promoterless lacZ reporter gene on a multicopy plasmid and selected 105 clones at random. Among the randomly selected clones, 56 had significantly elevated activity. Of these, 49 bore inserts that mapped within or downstream of ORFs. For six of the nine most active sequences with orientations opposite to that of the ORF, we detected chromosomal expression by RT-PCR but no defined transcripts by northern analysis. While our approach did not result in the identification of new small RNAs, the results did show that the E. coli chromosome carries numerous --35 and --10 sequences with weak promoter activity.

Development of general approaches for the characterization of small, noncoding RNAs

Opdyke, Zhang, Storz; in collaboration with Gottesman, Leppla, Tjaden

We have also 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 these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody. Subsequent reactions with a fluorescently labeled antimouse IgG antibody or biotin-labeled antimouse IgG together with fluorescently labeled streptavidin produce a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect 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 basepairing with target mRNAs. While the number of characterized small RNAs has steadily increased, only a limited number of the corresponding mRNA targets have been identified. In collaboration with Susan Gottesman and Brian Tjaden, we developed and tested TargetRNA, a program 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.

Characterization of specific small, noncoding RNAs

Fozo, Kawano, Opdyke, Zhang, Storz; in collaboration with Altuvia, Gottesman, Wassarman

Increasingly, our laboratory has been focusing on 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 basepairing with target mRNAs. OxyS RNA action is dependent on the Sm-like 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. Recently, we elucidated the functions of two other small RNAs that bind to Hfq and act by basepairing: the 109-nucleotide MicC RNA and the 105-nucleotide GadY RNA. 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. Thus, we suggest that the MicF and MicC RNAs act to control the OmpF:OmpC protein ratio in response to a variety of environmental stimuli. In contrast, basepairing between the GadY RNA and the 3′-untranslated region (3′ UTR) of the gadX mRNA encoded opposite gadY, leading 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. Studies are under way to characterize further the GadY RNA and the roles of other newly discovered small RNAs.

Characterization of small ORFs

Hemm, Miranda Rios, Paul, Soltanzad, Storz

In our genome-wide screens for small RNAs, we found that several short RNAs encode small proteins. Although small proteins have largely been missed, the few small proteins that have been studied in detail in bacterial and mammalian cells have been shown to have important functions in both signaling and cellular defenses. Thus, we established a project to elucidate the functions of E. coli proteins of less than 50 amino acids, using many of the approaches the group has used to characterize the functions of small, noncoding RNAs.

COLLABORATORS

Shoshy Altuvia, PhD, Hebrew University-Hadassah Medical School, Jerusalem, Israel
Susan Gottesman, PhD, Laboratory of Molecular Biology, NCI, Bethesda, MD
Stephen H. Leppla, PhD, Bacterial Toxins and Therapeutics Section, NIAID, Bethesda, MD
Robert G. Martin, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Judah L. Rosner, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, 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.