While redox-sensitive transcription factors and the bacterial and yeast responses to oxidative stress were a major focus for the group for many years, our attention has recently switched the identification and characterization of small, noncoding RNAs and small ORFs.
Characterization of small, noncoding RNAs
Small, noncoding RNA genes are of interest because, although these RNAs have largely been overlooked until recently, there is accumulating evidence that many act as important regulators in the cell. Noncoding RNA genes generally are not annotated and are poor targets in genetic screens. Noncoding RNAs also are often missed by biochemical approaches. Thus we have been carrying out systematic screens for additional noncoding RNA genes in E. coli. These 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 noncoding RNAs. Another screen for noncoding RNAs that coimmunoprecipitate with the RNA binding protein Hfq allowed us to detect six less well conserved RNAs. A third approach of size fraction of total RNA followed by linker ligation and cDNA synthesis led to the cloning of cis-encoded antisense RNAs.
The group has elucidated the functions of several of the noncoding RNAs in E. coli. We showed that the OxyS RNA, whose expression is induced 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 and modifies RNA polymerase. Recently, we found that the MicC RNA represses translation of the OmpC outer membrane porin. Interestingly, under most conditions, the MicC RNA shows the opposite expression as 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, we showed that basepairing 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. Studies to further characterize Hfq, the GadY RNA and the roles of other newly-discovered noncoding RNAs are ongoing.
Characterization of small ORFs
In our genome-wide screens for noncoding RNAs, we found that a number of short RNAs actually encode small proteins. Small proteins also have largely been overlooked in genome annotation and are not detected by many biochemical approaches. However, the few small proteins that have been studied in detail in bacterial and mammalian cells have been shown to have important functions in signaling and in cellular defenses. Thus we have recently initiated a project to elucidate the functions of E. coli proteins of less than 50 amino acids using many of the approaches we have used to characterize the functions of small, noncoding RNAs.