Small Regulatory Proteins

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications & trials

Abstract

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 proteins is one of the biggest 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 (1). 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 model Escherichia 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 also carried out a complementary approach based on genome-wide ribosome profiling of ribosomes arrested on start codons to identify many additional candidates; the synthesis of 38 of these small proteins was confirmed by chromosomal tagging. These studies together with the work of others has documented that E. coli synthesize over 150 small proteins. 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. Interestingly, 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 are using the tagged derivatives and information about synthesis and subcellular localization, along with 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. For example, we discovered the 49-amino acid inner membrane protein AcrZ, whose synthesis is increased in response to noxious compounds such as antibiotics and oxidizing agents, associates with the inner membrane AcrB component of the AcrAB-TolC multidrug efflux pump. Mutants lacking AcrZ are sensitive to many, but not all, of the antibiotics transported by AcrAB-TolC due to 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 by the MntR transcription factor. The lack of MntS leads to decreased 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 by 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 a 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. The ribosome profiling used to identify the intergenic-encoded small proteins revealed there is significant translation initiation within larger open reading frames in the E. coli genome. All five E. coli genes encoding Rpn (Recombination-promoting nuclease) proteins have such an internal translation site. We showed that the small, highly variable Rpn C-terminal domains (RpnS), which are translated separately from the full-length proteins (RpnL), directly block the activities of the toxic full-length RpnL proteins, comprising a novel toxin-antitoxin system (2). The crystal structure of RpnAS revealed a dimerization interface encompassing helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we documented that plasmid-encoded RpnP2L protects E. coli against certain phages. We propose that many more intragenic-encoded small proteins that serve regulatory roles remain to be discovered in all organisms. The ribosome profiling also revealed that some regulatory RNAs also encode a small protein and are thus dual-function RNAs. We documented 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) (3). Previous studies showed that overexpression of Spot 42 reduces growth in galactose. Overexpression of just the small protein from a Spot 42 derivative deficient in base-pairing activity resulted in the same phenotype, indicating that the sRNA and protein impact the same pathway. Co-purification experiments revealed that SpfP binds the global transcriptional regulator CRP. This binding blocks the ability of CRP to activate specific genes, impacting the kinetics of induction when cells are shifted from glucose to galactose medium. Thus, the small protein reinforces the feedforward loop regulated by the base-pairing activity of the Spot 42 RNA. Another 164-nucleotide RNA was previously shown to encode a 28-amino acid, amphipathic-helix protein (denoted AzuC). We discovered the membrane-associated AzuC protein interacts with GlpD, the aerobic glycerol-3-phosphate dehydrogenase, and increases dehydrogenase activity (4). Overexpression of the RNA encoding AzuC results in a growth defect in glycerol and galactose medium. The defect in galactose medium was still observed for a stop codon mutant derivative, suggesting a second role for the RNA. Consistent with this hypothesis, we found that cadA and galE are repressed by base pairing with the RNA (denoted AzuR) documenting that AzuCR is also 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 comprised of MgrR and MgtS (5). These constructs allowed us to probed 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, along with related findings by others in eukaryotic cells, supports our hypothesis that small proteins are an overlooked but important class of proteins, which we continue to study.