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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
• Aisha Burton Okala, PhD, Postdoctoral Fellow
• Sahar S. Melamed, PhD, Postdoctoral Fellow
• Medha V. Raina, PhD, Postdoctoral Fellow
• Narumon Thongdee, PhD, Postdoctoral Fellow
• Lauren R. Walling, PhD, Postdoctoral Fellow
• Mona Wu Orr, PhD, Postdoctoral Fellow
• Rilee D. Zeinert, PhD, Postdoctoral Fellow
• Aoshu Zhong, PhD, Postdoctoral Fellow
• Jordan J. Aoyama, MD, Graduate Student
• Abigail M. Daniels, MS, Postbaccalaureate Fellow
• Katherine M. Klier, BS, Postbaccalaureate Fellow
• Franchesca Uribe-Rheinbolt, BS, Postbaccalaureate Fellow
• Maxime Zamba-Campero, 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 RNAs (sRNAs) in Escherichia coli, which showed that sRNAs are encoded by diverse loci including the 5′ and 3′ UTRs of mRNAs [Reference 1]. 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. We are currently examining sRNA expression using several deep-sequencing approaches to further extend our identification of sRNAs in a range of bacteria species.

A major focus for the group has been to elucidate the functions of the sRNAs 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 depends on the Sm–like Hfq protein, which acts as a chaperone to facilitate OxyS RNA base pairing with its target mRNAs. We also started to explore the role of ProQ, a second RNA chaperone in E. coli. As shown in Figure 1, by comparing the sRNA–mRNA interactomes by deep sequencing, we found that ProQ and Hfq have overlapping as well as competing roles in the cell [Reference 2].

It is clear that Hfq–binding sRNAs, 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 3]. 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 a Sigma(E)–dependent sRNA, 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 from elevated Lpp levels. This observation raises the possibility that other phenotypes currently attributed to protein defects are the result of deficiencies in unappreciated regulatory RNAs. Studies to determine the factors that direct the cleavage of MicL, and likely other sRNAs, from the 3′ untranslated regions (UTRs) of mRNAs, showed that 3′ stem-loops are critical for the very specific processing [Reference 4]. Most recently while characterizing the response to limited magnesium, we found that the adjacently encoded MgrR sRNA and MgtS small protein both down regulate the pitA–encoded cation-phosphate symporter to increase intracellular magnesium levels [Reference 5].

In addition to sRNAs 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 another study, we discovered that a broadly conserved RNA structure motif yybP-ykoY motif, found in the 5′-UTR of the mntP gene encoding 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 sRNAs, we found that several short transcripts do indeed encode small proteins. The correct annotation of the genes encoding the smallest proteins is one of the biggest challenges of genome annotation. However, even though small-protein genes have been largely missed, the few small proteins studied in bacterial and mammalian cells have been shown to have important functions in regulation, signaling, and cellular defenses [Reference 6]. We thus established a project to identify and characterize proteins of less 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. The approach confirmed that 20 previously annotated and 18 newly discovered proteins of 16–50 amino acids are synthesized. We recently carried out a complementary approach based on genome-wide ribosome profiling of ribosomes arrested in start codons to identify many additional candidates; we confirmed the synthesis of 38 of these small proteins by chromosomal tagging. These studies, together with the work of others, documented that E. coli synthesizes over 150 small proteins.

Many of the initially discovered proteins were predicted to consist of a single transmembrane alpha-helix and, in biochemical fractionation, were found to be in the inner membrane. 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, to elucidate the functions of the small proteins, are employing many of the approaches the group used to characterize the functions of sRNAs. 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, is repressed by high levels of manganese. Correspondingly, 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. We also showed that the 31–amino acid inner-membrane protein MgtS (formerly denoted YneM), whose synthesis is induced by very low magnesium, 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. Most recently 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 [Reference 5].

We also 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 the interaction occurs through the inner-membrane protein AcrB. Mutants lacking AcrZ are sensitive to many, but not all, the antibiotics transported by AcrAB–TolC. Such 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 affects AcrB, and recently showed that AcrZ and cardiolipin cooperate to allosterically modulate AcrB activity.

This 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 important regulators.

Publications

1. Adams PP, Storz G. Prevalence of small base-pairing RNAs derived from diverse genomic loci. Biochim Biophys Acta Gene Regul Mech 2020;1863:194524.
2. Melamed S, Adams PP, Zhang A, Zhang H, Storz G. RNA-RNA interactomes of ProQ and Hfq reveal overlapping and competing roles. Mol Cell 2020;77:411-425.e7.
3. Hör J, Matera G, Vogel J, Gottesman S, Storz G. Trans-acting small RNAs and their effects on gene expression in Escherichia coli and Salmonella enterica. EcoSal Plus 2020;ESP-0030-2019.
4. Updegrove TB, Kouse AB, Bandyra KJ, Storz G. Stem-loops direct precise processing of 3' UTR-derived small RNA MicL. Nucleic Acids Res 2019;47:1482-1492.
5. Yin X, Wu Orr M, Wang H, Hobbs EC, Shabalina SA, Storz G. The small protein MgtS and small RNA MgrR modulate the PitA phosphate symporter to boost intracellular magnesium levels. Mol Microbiol 2019;111:131-144.
6. Hemm MR, Weaver J, Storz G. Escherichia coli small proteome. EcoSal Plus 2020;ESP-0031-2019.

Collaborators

• Katarzyna J. Bandyra, PhD, University of Cambridge, Cambridge, United Kingdom
• Allen R. Buskirk, PhD, Johns Hopkins Medical Institute, Baltimore, MD
• Dijun Du, PhD, School of Life Science and Technology, ShanghaiTech University, Pudong, China
• Susan Gottesman, PhD, Laboratory of Molecular Biology, Center for Cancer Research, NCI, Bethesda, MD
• Matthew R. Hemm, PhD, Department of Biological Sciences, Towson University, Towson, MD
• Syma Khalid, PhD, School of Chemistry, University of Southampton, Southampton, United Kingdom
• Ben F. Luisi, PhD, Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
• Shu-Bing Qian, PhD, Cornell University, Ithaca, NY
• Svetlana A. Shabalina, PhD, National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD
• Jörg Vogel, Dr rer nat, Institute of Molecular Infection Biology, Universität Würzburg, Würzburg, Germany
• Henry Zhang, PhD, Bioinformatics and Scientific Programming Core, NICHD, Bethesda, MD

Contact

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

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