GadY is unique in several ways with respect to other bacterial small RNAs. GadY is the first example of a bacterial small RNA that can form base pairs with the 3′ end of its target mRNA. Almost all of the characterized Hfq binding small RNAs modulate translation by forming base pairs with sequences spanning and adjacent to the ribosome binding site at the 5′ end of the target mRNA. In general, little is known about the 3′ UTRs of bacterial mRNAs and the type of regulatory signals they contain. More has been learned about regulation at the 3′ UTRs of eukaryotic transcripts, and several microRNAs in eukaryotic organisms have been shown to form base pairs within the 3′ UTRs of their target mRNAs (reviewed in reference 23
). We suggest that the formation of base pairs between small RNAs and 3′ UTRs will likely be found for other RNAs. It is worth noting that the gadY
gene arrangement couples the GadY small RNA to its target. Any mutations that occur within the pairing region will create a compensatory change on the opposite strand, with no net loss in complementarity between the small RNA and its mRNA target.
The GadY RNA is also the first E. coli small RNA that positively affects the accumulation of its target mRNA. So far, positive regulation has proved to be rare among RNA regulators. Only two small RNAs, DsrA and RprA of E. coli, have been found to positively regulate gene expression, and both of these function by enhancing translation of the rpoS target mRNA. Currently, no eukaryotic microRNAs are known to positively regulate gene expression, although it is conceivable that microRNAs with functions similar to GadY will be found.
The regulation of the genes involved in the glutamate-dependent acid response is complex and poorly understood. Control of this system has been shown to differ depending on the culture conditions (i.e., complex medium or minimal medium), and it involves multiple regulators (13
). Under the conditions we used for our studies, specifically growth in LB broth at pH 7.0, gadX
mRNA is expressed during stationary phase in a manner that is dependent on the secondary sigma factor σS
). We have shown that GadY transcription is also dependent on σS
. It was appealing to suggest that the gadX
dependence on σS
was mediated by GadY. We tested this possibility by overexpressing GadY in an rpoS
mutant and found that the gadX
mRNA did not accumulate in the rpoS
mutant strain that overexpressed GadY (data not shown). This suggests that gadX
mRNA accumulation during stationary phase is dependent on σS
for transcription initiation as well as on stabilization via the GadY RNA.
We have not ruled out the possibility that GadY regulates multiple targets, some of which are encoded at separate locations of the chromosome. Protein gel analysis indicated that the expression of at least four proteins is directly or indirectly affected by this small RNA. Intriguingly, the TnaA protein, which displays GadY-dependent repression, has been implicated in protecting cells against alkaline stress (4
). GadY targets could be regulated by all three forms of the RNA or could require one specific form of the GadY small RNA. Our GadY expression data suggest that the processing or stability of the three GadY small RNAs is different, as indicated by the fact that the 90-nucleotide form of the RNA persisted in late stationary phase while the full-length and 59-nucleotide forms became undetectable. If different forms of the small RNA are required for the regulation of individual target genes, it is possible that the targets are differentially regulated relative to each other.
GadY was shown to efficiently bind Hfq, an RNA binding protein required for the function of all small RNAs that form base pairs with target mRNAs. Although the mechanism of Hfq function is currently not fully understood, in vitro data for the OxyS and Spot42 small RNAs demonstrated that Hfq stimulates base pairing with the target mRNA (21
). Current models of Hfq function indicate that the protein may help to unfold regions of base pairing, increase local concentrations of the small RNA and its target, or both. These proposed activities are believed to be essential because base pairing between most trans
-encoded (encoded at a different location of the chromosome) small RNAs and their target mRNAs is short and noncontiguous. The GadY RNA is cis
-encoded (encoded on the opposite strand from the target), so by definition there is 100% complementarity between the small RNA and its target. The question arises regarding whether Hfq is required for GadY base pairing with the gadX
mRNA. It is possible that because of the extensive nature of the base pairing, Hfq is not needed to promote these interactions. This question is difficult to address, however, since GadY RNA levels were severely reduced in the Hfq mutant. Even if Hfq is not required for GadY base pairing with the gadX
mRNA, it may play a role in promoting base pairing with an unknown trans
GadY represents a novel mechanism by which a small RNA can positively affect the accumulation of its target, in this case the gadX
mRNA. We suggest that base pairing between the GadY small RNA and the gadX
mRNA protects the mRNA from degradation by an RNase. This mechanism would be the opposite to the one used by the RyhB small RNA to regulate iron storage proteins. RyhB functions by base pairing with specific mRNAs and rendering the duplex RNA sensitive to RNase E degradation (18
). Base pairing between GadY and the gadX
mRNA could impede the binding or activity of an RNase that functions at the 3′ end of the gadX
mRNA. E. coli
encodes two exoribonucleases, polynucleotidephosphorylase and RNase II, whose activities can be blocked by secondary structures that create double-stranded RNA (7
). Due to the fact that gadX
does not have an identifiable Rho-independent terminator comprised of a stem-loop, the unstructured 3′ end of the gadX
mRNA could be particularly sensitive to this type of RNase activity. The base pairing of GadY with the 3′ UTR of gadX
mRNA would create a double-stranded RNA duplex that could block the activity of such an RNase. Future experiments will be aimed at elucidating the mechanism of GadY protection of the gadX
mRNA as well as determining whether other small RNAs act similarly to stabilize their target mRNAs.