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Regulatory Small RNAs and Small Proteins

• Gisela Storz, PhD, Head, Section on Environmental Gene Regulation
• Aixia Zhang, PhD, Staff Scientist
• Jordan J. Aoyama, MD, Postdoctoral Fellow
• Aisha Burton Okala, PhD, Postdoctoral Fellow
• Rajat Dhyani, PhD, Postdoctoral Fellow
• Shuwen Shan, PhD, Postdoctoral Fellow
• Narumon Thongdee, PhD, Postdoctoral Fellow
• Lauren R. Walling, PhD, Postdoctoral Fellow
• Rilee D. Zeinert, PhD, Postdoctoral Fellow
• Aoshu Zhong, PhD, Postdoctoral Fellow
• Miranda Alaniz, BS, Postbaccalaureate Fellow
• Juwaan Douglas-Jenkins, BS, Postbaccalaureate Fellow
• Kyle D. Rekedal, BS, Postbaccalaureate Fellow
• Tiara D. Tillis, BA, Postbaccalaureate Fellow

The group currently has two main interests: identification and characterization of small noncoding RNAs; and 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 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 RNAs in Escherichia coli. The screens included computational searches for conservation of intergenic regions and direct detection after size selection or co-immunoprecipitation with RNA–binding proteins. Most recently, we have been using deep sequencing approaches to map the 5′ and 3′ ends of all transcripts to further extend our identification of small RNAs in a range of bacteria species [Reference 1]. The work showed that sRNAs are encoded by diverse loci, including sequences overlapping mRNAs.

A major focus for the group has been to elucidate the functions of the small RNAs that 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. Follow up studies allowed us to learn more about the mechanism by which the Hfq protein facilitates base-pairing through multiple RNA binding domains [Reference 2]. We also started to explore the role of ProQ, a second RNA chaperone in E. coli and, by comparing the sRNA–mRNA interactomes by deep sequencing, found that ProQ and Hfq have overlapping as well as competing roles in the cell. It is likely that still other RNA binding proteins, such as KH domain proteins, are involved in small RNA–mediated regulation in bacteria [Reference 3].

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. 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 MicL, a transcription factor Sigma(E)–dependent small RNA, which is 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. Most recently, we showed that a small RNA derived from the 3′ UTR (untranslated region) of the glnA gene, encoding glutamine synthetase, impacts E. coli growth under low nitrogen conditions by modulating the expression of genes that affect carbon and nitrogen flux [Reference 4]. As more and more sRNAs encoded by 5′ or 3′ UTRs or internal to coding sequences are being found, our observations raise the possibility that other phenotypes currently attributed to protein defects are the result of deficiencies in previously unidentified regulatory RNAs.

One interesting recent observation is that some small RNAs have dual functions in that they act by both base-pairing and by encoding a small, regulatory protein. For example, we discovered that the Spot 42 RNA also encodes a 15–amino acid protein (denoted SpfP) [Reference 5]. Overexpression of just the small protein from a Spot 42 derivative deficient in base-pairing activity, or just the base-pairing activity from a Spot 42 derivative with a stop codon mutation both prevented growth on galactose, revealing that the small protein and the small RNA impact the same pathway. Co-purification experiments showed that SpfP binds to the CRP (cyclic AMP receptor protein) transcription factor, affecting the kinetics of induction when cells are shifted from a glucose to a galactose medium. Thus, as shown in Figure 1, the small protein reinforces the feedforward loop regulated by the base-pairing activity of the Spot 42 RNA. As a second example, we found a 164–nucleotide RNA previously shown to encode a 28–amino acid protein (denoted AzuC) also base-pairs with the cadA and galE mRNAs to block expression [Reference 6]. Interestingly, AzuC translation interferes with the observed repression of cadA and galE by the RNA, and base-pairing interferes with AzuC translation, demonstrating that the translation and base-pairing functions compete. We hypothesize that many more dual-function RNAs remain to be discovered and suggest that they can be exploited to control gene expression at many levels. We successfully constructed a functional synthetic dual-function regulator from a small protein and a small protein encoded by adjacent genes, and used this synthetic construct to study the functional organization of dual-function RNAs [Aoyama JJ, Raina M, Storz G. J Bacteriol 2022;204:e00345–21].

In addition to small RNAs that act via limited base pairing, we have been interested in regulatory RNAs that act by other mechanisms. For instance, early work showed that the 6S RNA binds to and modulates RNA polymerase by mimicking the structure of an open promoter. In another study, we discovered that a broadly conserved RNA structure motif, the yybP-ykoY motif, found in the 5′ UTR of the mntP gene, which encodes a manganese exporter, directly binds manganese, resulting in a conformation that liberates the ribosome-binding site.

Further studies to characterize other Hfq– and ProQ–binding RNAs and their physiological roles and evolution as well as regulatory RNAs that 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 actually encode small proteins. The correct annotation of the smallest protein genes is one of the greatest challenges of genome annotation. Further, there is limited evidence that proteins are synthesized from annotated and predicted short ORFs. Although these 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 regulation, signaling, and in cellular defenses [Gray T, Storz G, Papenfort K. J Bacteriol 2022;204:e0034121]. We thus established a project to identify and characterize proteins of fewer than 50 amino acids.

We first 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 to 50 amino acids are synthesized. We also carried out a complementary approach, based on genome-wide ribosome profiling of ribosomes arrested on start codons, to identify many additional candidates; we confirmed the synthesis of 38 of these small proteins by chromosomal tagging. Our studies, together with the work of others, documented that E. coli synthesizes over 150 small proteins of fewer than 50 amino acids.

Many of the initially discovered proteins were predicted to consist of a single transmembrane alpha-helix and were found to be in the inner membrane in biochemical fractionation. 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.

We now are using the tagged derivatives and information about synthesis and subcellular localization, and are employing many of the approaches the group has used to characterize the functions of small regulatory RNAs, to elucidate the functions of the small proteins. The combined approaches are beginning to give insights into how the small proteins are acting in E. coli. We first discovered that the 49–amino acid inner-membrane protein AcrZ, whose synthesis increases 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, 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, resulting from AcrZ effects on the conformation of the AcrB drug–binding pocket. We also found that synthesis of a 42–amino acid protein, MntS, is repressed by high levels of manganese and the MntR transcription factor. The lack of MntS leads to reduced activities of manganese-dependent enzymes under manganese-poor conditions, while overproduction of MntS leads to very high intracellular manganese and bacteriostasis under manganese-rich conditions. These and other phenotypes led us to propose that MntS modulates intracellular manganese levels, possibly by inhibiting the manganese exporter MntP. Additionally, we showed that the 31–amino acid inner-membrane protein MgtS, whose synthesis is induced by very low magnesium and the PhoPQ two-component system, acts to increase intracellular magnesium levels and maintain cell integrity upon magnesium depletion. Upon development of a functional tagged derivative of MgtS, we found that MgtS interacts with MgtA to increase the levels of this P-type ATPase magnesium transporter under magnesium-limiting conditions. Correspondingly, the effects of MgtS upon magnesium limitation are lost in an mgtA mutant, and MgtA overexpression can suppress the mgtS phenotype. MgtS stabilization of MgtA provides an additional layer of regulation of this tightly controlled magnesium transporter. Unexpectedly, we found that MgtS also interacts with and modulates the activity of a second protein, the PitA cation–phosphate symporter, to further increase intracellular magnesium levels.

A limited number of transcripts encoding both a small protein and possessing base-pairing activity, dual-function RNAs, are being identified. For example, the 109–nucleotide Spot 42 RNA, which is one of the best characterized base-pairing small RNAs (sRNAs) in E. coli encodes a 15–amino acid protein (denoted SpfP). As mentioned above, overexpression of just the Spot 42 base-pairing activity or just the SpfP small protein prevented growth on galactose, indicating that the sRNA and protein impact the same pathway [Reference 5]. SpfP binding to CRP blocks the ability of the transcription factor to activate specific genes, reinforcing the feedforward loop regulated by the base-pairing activity of the Spot 42 RNA (Figure 1). A second example is the 164–nucleotide RNA previously shown to encode a 28–amino acid, amphipathic-helix protein (denoted AzuC). We discovered that the membrane-associated AzuC protein interacts with GlpD, the aerobic glycerol-3-phosphate dehydrogenase, and increases dehydrogenase activity [Reference 6]. The observations that an overexpression defect was still observed for a stop-codon mutant derivative and that the RNA (denoted AzuR) can also base-pair with the cadA and galE mRNAs documented that AzuCR is a dual-function RNA. Interestingly, the MgtS protein mentioned above is encoded divergent from the MgrR small regulatory RNA, which is also important for bacterial adaptation to low magnesium. To investigate the competition between protein-coding and base-pairing activities, we constructed synthetic dual-function RNAs comprising MgrR and MgtS [Aoyama JJ, Raina M, Storz G. J Bacteriol 2022;204:e00345–21]. The constructs allowed us to probe how the organization of the coding and base-pairing sequences and the distance between the two components contribute to the proper function of both activities of a dual-function RNA. By understanding the features of natural and synthetic dual-function RNAs, future synthetic molecules can be designed to maximize their regulatory impact.

Our work, together with our ongoing studies of other small proteins and related findings by others in eukaryotic cells, supports our hypothesis that small proteins are an overlooked class of proteins.

Additonal Funding

• NICHD Early Career Award
• NIGMS Postdoctoral Research Associate (PRAT) Program

Publications

1. Adams PP, Baniulyte G, Esnault C, Chegireddy K, Singh N, Monge M, Dale RK, Storz G, Wade JT. Regulatory roles of Escherichia coli 5´ UTR and ORF-internal RNAs detected by 3′ end mapping. eLife 2021 10:e62438.
2. Kavita K, Zhang A, Tai CH, Majdalani N, Storz G, Gottesman S. Multiple in vivo roles for the C-terminal domain of the RNA chaperone Hfq. Nucleic Acids Res 2022 50:1718–1733.
3. Olejniczak M, Jiang X, Basczok MM, Storz G. KH-domain proteins: another family of bacterial RNA matchmakers? Mol Microbiol 2022 117:10-19.
4. Walling LR, Kouse AB, Shabalina SA, Zhang H, Storz G. A 3′ UTR-derived small RNA connecting nitrogen and carbon metabolism in enteric bacteria. Nucleic Acids Res 2022 50:10093–10109.
5. Aoyama JJ, Raina M, Zhong A, Storz G. Dual-function Spot 42 RNA encodes a 15-amino acid protein that regulates the CRP transcription factor. Proc Natl Acad Sci USA 2022 119:e2119866119.
6. Raina M, Aoyama JJ, Bhatt S, Paul BJ, Zhang A, Updegrove TB, Miranda-Ríos J, Storz G. Dual-function AzuCR RNA modulates carbon metabolism. Proc Natl Acad Sci USA 2022 119:e2117930119.

Collaborators

• Shantanu Bhatt, PhD, Department of Biology, Saint Joseph's University, Philadelphia, PA
• Ryan K. Dale, PhD, Bioinformatics and Scientific Programming Core, NICHD, Bethesda, MD
• Caroline Esnault, PhD, Bioinformatics and Scientific Programming Core, NICHD, Bethesda, MD
• Susan Gottesman, PhD, Laboratory of Molecular Biology, Center for Cancer Research, NCI, Bethesda, MD
• Todd Gray, PhD, Wadsworth Center, New York State Department of Health, Albany, NY
• Xiaofang Jiang, PhD, National Library of Medicine, NIH, Bethesda, MD
• Nadim Majdalani, PhD, Laboratory of Molecular Biology, Center for Cancer Research, NCI, Bethesda, MD
• Juan Miranda-Rios, PhD, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico
• Mikolaj Olejniczak, PhD, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland
• Kai Papenfort, PhD, Institute of Microbiology, Friedrich-Schiller-Universität, Jena, Germany
• Brian J. Paul, PhD, International Flavors & Fragrances Inc., Wilmington, DE
• Svetlana A. Shabalina, PhD, National Library of Medicine, NIH, Bethesda, MD
• Chin-Hsien Tai, PhD, Laboratory of Molecular Biology, Center for Cancer Research, NCI, Bethesda, MD
• Taylor B. Updegrove, PhD, Laboratory of Molecular Biology, Center for Cancer Research, NCI, Bethesda, MD
• Joseph T. Wade, PhD, Wadsworth Center, New York State Department of Health, Albany, NY
• Henry Zhang, PhD, Bioinformatics and Scientific Programming Core, NICHD, Bethesda, MD

Contact

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

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