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Regulatory Small RNAs and Small Proteins

• Gisela Storz, PhD, Head, Section on Environmental Gene Regulation
• Aixia Zhang, PhD, Staff Scientist
• Philip P. Adams, PhD, Postdoctoral Fellow
• Andrew B. Kouse, PhD, Postdoctoral Fellow
• Sahar S. Melamed, PhD, Postdoctoral Fellow
• Medha V. Raina, PhD, Postdoctoral Fellow
• Lauren R. Walling, PhD, Postdoctoral Fellow
• Jeremy S. Weaver, PhD, Postdoctoral Fellow
• Mona Wu Orr, PhD, Postdoctoral Fellow
• Jordan J. Aoyama, MD, Graduate Student
• Joshua M. Mills, BS, Postbaccalaureate Fellow
• Leana M. Ramos, BS, Postbaccalaureate Fellow
• Franchesca Uribe-Rheinbolt, BS, Postbaccalaureate Fellow

The group currently has two main interests: (1) identification and characterization of small noncoding RNAs and (2) identification and characterization of small proteins of less than 50 amino acids. Both small RNAs and small proteins have been overlooked because they are not detected in biochemical assays and the corresponding genes are missed by genome annotation and are poor targets for genetic approaches. However, both classes of small molecules are now being found to have important regulatory roles in organisms ranging from bacteria to humans.

Identification and characterization of small regulatory RNAs

During the past 20 years, we have carried out several different systematic screens for small regulatory RNA genes in Escherichia coli. The screens included computational searches for conservation of intergenic regions and direct detection after size selection or co-immunoprecipitation with the RNA–binding protein Hfq. To further extend our identification of small RNAs in a range of bacteria species, we recently examined small RNA expression using deep sequencing.

A major focus for the group has been to elucidate the functions of the small RNAs we and others identified. Early on, we showed that the OxyS RNA, whose expression is induced in response to oxidative stress, acts to repress translation through limited base pairing with target mRNAs. We discovered that OxyS action is dependent on the Sm–like Hfq protein, which acts as a chaperone to facilitate OxyS RNA base pairing with its target mRNAs. We have now also started to explore the role of ProQ, a second RNA chaperone in E. coli and other bacteria [Reference 1].

It is clear that Hfq–binding small RNAs, which act through limited base pairing, are integral to many different stress responses in E. coli and other bacteria, as well as during the interaction between bacteria and bacteriophage [Reference 2]. For example, we showed that the Spot 42 RNA, whose levels are highest when glucose is present, plays a broad role in catabolite repression by directly repressing genes involved in central and secondary metabolism, redox balancing, and the consumption of diverse non-preferred carbon sources. Similarly, we discovered that the Sigma(E)-dependent small RNA MicL, transcribed from a promoter located within the coding sequence of the cutC gene, represses synthesis of the lipoprotein Lpp, the most abundant protein in the cell, to oppose membrane stress. We found that the copper-sensitivity phenotype previously ascribed to inactivation of the cutC gene is actually derived from the loss of MicL and elevated Lpp levels. Our observation raises the possibility that other phenotypes currently attributed to protein defects can rather be attributed to deficiencies in unappreciated regulatory RNAs. Most recently, we characterized a set of small RNAs expressed from a locus we named sdsN. Two longer sRNAs, SdsN137 and SdsN178, are transcribed from two Sigma(S)-dependent promoters but share the same terminator. Whole genome expression analysis, after pulse overexpression of SdsN137 and assays of lacZ fusions, revealed that SdsN137 directly represses the synthesis of the nitroreductase NfsA, which catalyzes the reduction of the nitrogroup (NO₂) in nitroaromatic compounds, and of the flavohemoglobin HmpA, which has aerobic nitric oxide (NO) dioxygenase activity. Consistent with this regulation, SdsN137 confers resistance to nitrofurans. Interestingly, SdsN178 is defective in regulating the above targets because of unusual binding to the Hfq protein, but cleavage leads to a shorter form, SdsN124, able to repress nfsA and hmpA.

In addition to small RNAs that act via limited base pairing, we have been interested in regulatory RNAs that act by other mechanisms. For example, early work showed that the 6S RNA binds to and modulates RNA polymerase by mimicking the structure of an open promoter. In a more recent study, we discovered that a broadly conserved RNA structure motif, the yybP-ykoY motif, found in the 5′ UTR of the E. coli mntP gene encoding a manganese (Mn²⁺) exporter, directly binds Mn²⁺, resulting in a conformation that liberates the ribosome-binding site. Remarkably, we were able to recapitulate the effect of Mn²⁺–dependent activation of translation in vitro. We also found that the yybP-ykoY motif responds directly to Mn²⁺ in Bacillus subtilis. The identification of the yybP-ykoY motif as a Mn²⁺ sensor suggests that the genes preceded by this motif, and which encode a diverse set of poorly characterized membrane proteins, have roles in metal homeostasis.

Further studies to characterize other Hfq–binding RNAs and their evolution as well as regulatory RNAs that bind to other proteins such as ProQ and act in ways other than base pairing are ongoing.

Identification and characterization of small proteins

In our genome-wide screens for small RNAs, we found that a number of short RNAs encode small proteins [Reference 3]. The correct annotation of the smallest proteins is one of the biggest challenges of genome annotation, and there is little evidence that annotated short open reading frames (ORFs) encode synthesized proteins. Although such 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 signaling and in cellular defenses. We thus established a project to identify and characterize proteins of less than 50 amino acids.

We used sequence conservation and ribosome binding–site models to predict genes encoding small proteins of 16–50 amino acids, in the intergenic regions of the E. coli genome. We tested expression of these predicted as well as previously annotated small proteins by integrating the sequential peptide affinity tag directly upstream of the stop codon on the chromosome and assaying for synthesis using immunoblot assays. This approach confirmed that 20 previously annotated and 18 newly discovered proteins of 16–50 amino acids are synthesized. We have now initiated a complementary approach, based on genome-wide ribosome profiling of ribosomes arrested in start codons, to identify additional small proteins.

More than half the newly discovered proteins were predicted to consist of a single transmembrane alpha-helix and, by biochemical fractionation, were found to be located in the inner membrane. Interestingly, assays of topology-reporter fusions and strains with defects in membrane insertion proteins revealed that, despite their diminutive size, small membrane proteins display considerable diversity in topology and insertion pathways. Additionally, systematic assays for the accumulation of tagged versions of the proteins showed that many small proteins accumulate under specific growth conditions or after exposure to stress. We also generated and screened bar-coded null mutants and identified small proteins required for resistance to cell-envelope stress and acid shock.

To elucidate the functions of the small proteins, we are now using the tagged derivatives and information about synthesis and subcellular localization and employing many of the approaches the group has used to characterize the functions of small regulatory RNAs. The combined approaches are beginning to yield insights into how the small proteins act in E. coli. We found that synthesis of a 42–amino acid protein, now denoted MntS (formerly the small RNA gene rybA), is repressed by high levels of Mn²⁺ through MntR. The lack of MntS leads to reduced activity of Mn²⁺-dependent enzymes under Mn²⁺-poor conditions, whereas overproduction of MntS leads to very high intracellular Mn²⁺ and bacteriostasis under Mn²⁺-rich conditions. These and other phenotypes led us to propose a model whereby, during transitions between low- and high-Mn²⁺environments, E. coli uses the Mn²⁺ exporter MntP to compensate for overactivity of the Mn²⁺ importer MntH and MntS to compensate for MntP overactivity.

We also showed that the 31–amino acid inner membrane protein MgtS (formerly denoted YneM), whose synthesis is induced by very low magnesium (Mg²⁺) by the PhoPQ two-component system in E. coli, acts to increase Mg²⁺ levels and maintain cell integrity upon Mg²⁺ depletion (Figure 1). Upon development of a functional tagged derivative of MgtS, we showed that MgtS interacts with MgtA to increase the levels of this P-type ATPase Mg²⁺ transporter under Mg²⁺-limiting conditions [Reference 4]. MgtS stabilization of MgtA provides an additional layer of regulation of this tightly controlled transporter. Surprisingly, we found that overexpression of the MgtS protein also leads to induction of the low-phosphate regulon controlled by the PhoRB two-component system. Studies to understand this activation showed that MgtS, although consisiting of only 31 amino acids, forms a complex with a second protein, PitA, a cation-phosphate symporter [Reference 5]. Given that the additive effects of deleting mgtA and mgtS on intracellular Mg²⁺ concentrations seen previously are lost in the pitA mutant, we suggest that MgtS binds to and prevents Mg²⁺ leakage through PitA under Mg²⁺-limiting conditions. Consistent with a previously unappreciated detrimental role of PitA in low Mg²⁺, we also observed MgrR sRNA repression of PitA synthesis. Thus, in response to Mg²⁺ limitation, PhoQP induces the expression of two convergent small genes whose products act to modulate PitA at different levels to increase intracellular Mg²⁺.

We discovered the 49–amino acid inner membrane protein AcrZ (formerly named YbhT), whose synthesis is increased in response to noxious compounds such as antibiotics and oxidizing agents, associates with the AcrAB–TolC multidrug efflux pump, which confers resistance to a wide variety of antibiotics and other compounds. Co-purification of AcrZ with AcrB (in the absence of both AcrA and TolC), two-hybrid assays, and suppressor mutations indicate that this interaction occurs through the inner membrane protein AcrB. Mutants lacking AcrZ are sensitive to many, but not all, the antibiotics transported by AcrAB–TolC. The differential antibiotic sensitivity suggests that AcrZ enhances the ability of the AcrAB–TolC pump to export certain classes of substrates. Detailed structural and mutational studies are now giving insight into how AcrZ changes AcrB activity.

This work, together with our ongoing studies on other small proteins and related findings by others in eukaryotic cells, support our hypothesis that many small proteins act as regulators of larger membrane proteins.

Additional Funding

• NICHD Director's Investigator Award

Publications

1. Updegrove TB, Zhang A, Storz G. Hfq: the flexible RNA matchmaker. Curr Opin Microbiol 2016 30:133-138.
2. Olejniczak M, Storz G. ProQ/FinO-domain proteins: another ubiquitous family of RNA matchmakers? Mol Microbiol 2017 104:905-915.
3. Machner MP, Storz G. Infection biology: small RNA with a large impact. Nature 2016 529:472-473.
4. Hao Y, Updegrove TB, Livingston NN, Storz G. Protection against deleterious nitrogen compounds: role of sigmaS-dependent small RNAs encoded adjacent to sdiA. Nucleic Acids Res 2016 44:6935-6948.
5. Storz G. New perspectives: insights into oxidative stress from bacterial studies. Arch Biochem Biophys 2016 595:25-27.
6. Wang H, Yin X, Wu Orr M, Dambach M, Curtis R, Storz G. Increasing intracellular magnesium levels with the 31-amino acid MgtS protein. Proc Natl Acad Sci USA 2017 114:5689-5694.
7. Raina M, Storz G. Sort, a small protein that packs a sweet punch. J Bacteriol 2017 199:e00130-17.

Collaborators

• Matthias P. Machner, PhD, Section on Microbial Pathogenesis, NICHD, Bethesda, MD
• Mikolaj Olejniczak, PhD, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland

Contact

For more information, email storz@helix.nih.gov or visit http://storz.nichd.nih.gov.

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