Small proteins can no longer be ignored.

Small proteins, here defined as proteins of 50 amino acids or fewer in the absence of processing, have traditionally been overlooked due to challenges in their annotation and biochemical detection. In the past several years, however, increasing numbers of small proteins have been identified either through the realization that mutations in intergenic regions are actually within unannotated small protein genes or through the discovery that some small, regulatory RNAs encode small proteins. These insights, together with comparative sequence analysis, indicate that tens if not hundreds of small proteins are synthesized in a given organism. This review summarizes what has been learned about the functions of several of these bacterial small proteins, most of which act at the membrane, illustrating the astonishing range of processes in which these small proteins act and suggesting several general conclusions. Important questions for future studies of these overlooked proteins are also discussed.

Large Roles of Small Proteins.

Bacterial proteins of ≤50 amino acids, denoted small proteins or microproteins, have been traditionally understudied and overlooked, as standard computational, biochemical, and genetic approaches often do not detect proteins of this size. However, with the realization that small proteins are stably expressed and have important cellular roles, there has been increased identification of small proteins in bacteria and eukaryotes. Gradually, the functions of a few of these small proteins are being elucidated. Many interact with larger protein products to modulate their subcellular localization, stabilities, or activities. Here, we provide an overview of these diverse functions in bacteria, highlighting generalities among bacterial small proteins and similarly sized proteins in eukaryotic organisms and discussing questions for future research.

RNA-RNA Interactomes of ProQ and Hfq Reveal Overlapping and Competing Roles.

Base pairing RNAs modulate gene expression in all studied organisms. In many bacteria, the base pairing between most small regulatory RNAs (sRNAs) and their targets is mediated by the Hfq RNA chaperone. However, recent studies have shown FinO-domain proteins also bind sRNAs. To examine the global contribution of the FinO-domain ProQ protein in Escherichia coli, we carried out RIL-seq to identify RNA pairs bound to this protein. The RNA-RNA interactome for ProQ contains hundreds of pairs. Intriguingly, a significant fraction of the ProQ-bound RNA pairs are also found associated with Hfq, indicating overlapping, complementary, or competing roles for the two proteins. Characterization of one novel RNA pair bound by both chaperones revealed that while Hfq is required for RNA sponge-mediated downregulation of the sRNA, ProQ can inhibit this regulation. Overall, our results uncover increased complexity in RNA regulatory networks involving RNA chaperone proteins, RNases, sRNAs, and mRNAs.

Two for one: regulatory RNAs that encode small proteins.

RNAs are commonly categorized as being either protein-coding mRNAs or noncoding RNAs. However, an increasing number of transcripts, in organisms ranging from bacteria to humans, are being found to have both coding and noncoding functions. In some cases, the sequences encoding the protein and the regulatory RNA functions are separated, while in other cases the sequences overlap. The protein and RNA can regulate similar or distinct pathways. Here we describe examples illustrating how these dual-function (also denoted bifunctional or dual-component) RNAs are identified and their mechanisms of action and cellular roles. We also discuss the synergy or competition between coding and RNA activity and how these regulators evolved, as well as how more dual-function RNAs might be discovered and exploited.

An acetyltranferase moonlights as a regulator of the RNA binding repertoire of the RNA chaperone Hfq in Escherichia coli.

Bacterial small RNAs (sRNAs) regulate gene expression by base-pairing with their target mRNAs. In Escherichia coli and many other bacteria, this process is dependent on the RNA chaperone Hfq, a mediator for sRNA-mRNA annealing. YhbS (renamed here as HqbA), a putative Gcn5-related N-acetyltransferase (GNAT), was previously identified as a silencer of sRNA signaling in a genomic library screen. Here, we studied how HqbA regulates sRNA signaling and investigated its physiological roles in modulating Hfq activity. Using fluorescent reporter assays, we found that HqbA overproduction suppressed all tested Hfq-dependent sRNA signaling. Direct interaction between HqbA and Hfq was demonstrated both in vivo and in vitro, and mutants that blocked the interaction interfered with HqbA suppression of Hfq. However, an acetylation-deficient HqbA mutant still disrupted sRNA signaling, and HqbA interacted with Hfq at a site far from the active site. This suggests that HqbA may be bifunctional, with separate roles for regulating via Hfq interaction and for acetylation of undefined substrates. Gel shift assays revealed that HqbA strongly reduced the interaction between the Hfq distal face and low-affinity RNAs but not high-affinity RNAs. Comparative RNA immunoprecipitation of Hfq and sequencing showed enrichment of two tRNA precursors, metZWV and proM, by Hfq in mutants that lost the HqbA-Hfq interaction. Our results suggest that HqbA provides a level of quality control for Hfq by competing with low-affinity RNA binders.

Toxic antiphage defense proteins inhibited by intragenic antitoxin proteins.

Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here, we report that these proteins are toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show 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 RpnL. The crystal structure of RpnAS revealed a dimerization interface encompassing α 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 document that plasmid-encoded RpnP2L protects Escherichia coli against certain phages. We propose that many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms.

Dual-function Spot 42 RNA encodes a 15-amino acid protein that regulates the CRP transcription factor.

SignificanceDual-function RNAs base pair with target messenger RNAs as small regulatory RNAs and encode small protein regulators. However, only a limited number of these dual-function regulators have been identified. In this study, we show that a well-characterized base-pairing small RNA surprisingly also encodes a 15-amino acid protein. The very small protein binds the cyclic adenosine monophosphate receptor protein transcription factor to block activation of some promoters, raising the question of how many other transcription factors are modulated by unidentified small proteins.

Dual-function AzuCR RNA modulates carbon metabolism.

SignificanceWhile most small, regulatory RNAs are thought to be "noncoding," a few have been found to also encode a small protein. Here we describe a 164-nucleotide RNA that encodes a 28-amino acid, amphipathic protein, which interacts with aerobic glycerol-3-phosphate dehydrogenase and increases dehydrogenase activity but also base pairs with two mRNAs to reduce expression. The coding and base-pairing sequences overlap, and the two regulatory functions compete.

Regulatory RNAs in bacteria.

Bacteria possess numerous and diverse means of gene regulation using RNA molecules, including mRNA leaders that affect expression in cis, small RNAs that bind to proteins or base pair with target RNAs, and CRISPR RNAs that inhibit the uptake of foreign DNA. Although examples of RNA regulators have been known for decades in bacteria, we are only now coming to a full appreciation of their importance and prevalence. Here, we review the known mechanisms and roles of regulatory RNAs, highlight emerging themes, and discuss remaining questions.

Multiple in vivo roles for the C-terminal domain of the RNA chaperone Hfq.

Hfq, a bacterial RNA chaperone, stabilizes small regulatory RNAs (sRNAs) and facilitates sRNA base-pairing with target mRNAs. Hfq has a conserved N-terminal domain and a poorly conserved disordered C-terminal domain (CTD). In a transcriptome-wide examination of the effects of a chromosomal CTD deletion (Hfq1-65), the Escherichia coli mutant was most defective for the accumulation of sRNAs that bind the proximal and distal faces of Hfq (Class II sRNAs), but other sRNAs also were affected. There were only modest effects on the levels of mRNAs, suggesting little disruption of sRNA-dependent regulation. However, cells expressing Hfq lacking the CTD in combination with a weak distal face mutation were defective for the function of the Class II sRNA ChiX and repression of mutS, both dependent upon distal face RNA binding. Loss of the region between amino acids 66-72 was critical for this defect. The CTD region beyond amino acid 72 was not necessary for distal face-dependent regulation, but was needed for functions associated with the Hfq rim, seen most clearly in combination with a rim mutant. Our results suggest that the C-terminus collaborates in various ways with different binding faces of Hfq, leading to distinct outcomes for individual sRNAs.

A 3' UTR-derived small RNA connecting nitrogen and carbon metabolism in enteric bacteria.

Increasing numbers of small, regulatory RNAs (sRNAs) corresponding to 3' untranslated regions (UTR) are being discovered in bacteria. One such sRNA, denoted GlnZ, corresponds to the 3' UTR of the Escherichia coli glnA mRNA encoding glutamine synthetase. Several forms of GlnZ, processed from the glnA mRNA, are detected in cells growing with limiting ammonium. GlnZ levels are regulated transcriptionally by the NtrC transcription factor and post-transcriptionally by RNase III. Consistent with the expression, E. coli cells lacking glnZ show delayed outgrowth from nitrogen starvation compared to wild type cells. Transcriptome-wide RNA-RNA interactome datasets indicated that GlnZ binds to multiple target RNAs. Immunoblots and assays of fusions confirmed GlnZ-mediated repression of glnP and sucA, encoding proteins that contribute to glutamine transport and the citric acid cycle, respectively. Although the overall sequences of GlnZ from E. coli K-12, Enterohemorrhagic E. coli and Salmonella enterica have significant differences due to various sequence insertions, all forms of the sRNA were able to regulate the two targets characterized. Together our data show that GlnZ impacts growth of E. coli under low nitrogen conditions by modulating genes that affect carbon and nitrogen flux.

KH domain proteins: Another family of bacterial RNA matchmakers?

In many bacteria, the stabilities and functions of small regulatory RNAs (sRNAs) that act by base pairing with target RNAs most often are dependent on Hfq or ProQ/FinO-domain proteins, two classes of RNA chaperone proteins. However, while all bacteria appear to have sRNAs, many have neither Hfq nor ProQ/FinO-domain proteins raising the question of whether another factor might act as an sRNA chaperone in these organisms. Several recent studies have reported that KH domain proteins, such as KhpA and KhpB, bind sRNAs. Here we describe what is known about the distribution, structures, RNA-binding properties, and physiologic roles of KhpA and KhpB and discuss evidence for and against these proteins serving as sRNAs chaperones.

The ubiquitous yybP-ykoY riboswitch is a manganese-responsive regulatory element.

The highly structured, cis-encoded RNA elements known as riboswitches modify gene expression upon binding a wide range of molecules. The yybP-ykoY motif was one of the most broadly distributed and numerous bacterial riboswitches for which the cognate ligand was unknown. Using a combination of in vivo reporter and in vitro expression assays, equilibrium dialysis, and northern analysis, we show that the yybP-ykoY motif responds directly to manganese ions in both Escherichia coli and Bacillus subtilis. The identification of the yybP-ykoY motif as a manganese ion sensor suggests that the genes that are preceded by this motif and encode a diverse set of poorly characterized membrane proteins have roles in metal homeostasis.

Small Proteins; Big Questions.

In recent years, there has been increased appreciation that a whole category of proteins, small proteins of around 50 amino acids or fewer in length, has been missed by annotation as well as by genetic and biochemical assays. With the increased recognition that small proteins are stable within cells and have regulatory functions, there has been intensified study of these proteins. As a result, important questions about small proteins in bacteria and archaea are coming to the fore. Here, we give an overview of these questions, the initial answers, and the approaches needed to address these questions more fully. More detailed discussions of how small proteins can be identified by ribosome profiling and mass spectrometry approaches are provided by two accompanying reviews (N. Vazquez-Laslop, C. M. Sharma, A. S. Mankin, and A. R. Buskirk, J Bacteriol 204:e00294-21, 2022, https://doi.org/10.1128/JB.00294-21; C. H. Ahrens, J. T. Wade, M. M. Champion, and J. D. Langer, J Bacteriol 204:e00353-21, 2022, https://doi.org/10.1128/JB.00353-21). We are excited by the prospects of new insights and possible therapeutic approaches coming from this emerging field.

Alternative ORFs and small ORFs: shedding light on the dark proteome.

Traditional annotation of protein-encoding genes relied on assumptions, such as one open reading frame (ORF) encodes one protein and minimal lengths for translated proteins. With the serendipitous discoveries of translated ORFs encoded upstream and downstream of annotated ORFs, from alternative start sites nested within annotated ORFs and from RNAs previously considered noncoding, it is becoming clear that these initial assumptions are incorrect. The findings have led to the realization that genetic information is more densely coded and that the proteome is more complex than previously anticipated. As such, interest in the identification and characterization of the previously ignored 'dark proteome' is increasing, though we note that research in eukaryotes and bacteria has largely progressed in isolation. To bridge this gap and illustrate exciting findings emerging from studies of the dark proteome, we highlight recent advances in both eukaryotic and bacterial cells. We discuss progress in the detection of alternative ORFs as well as in the understanding of functions and the regulation of their expression and posit questions for future work.

MicL, a new σE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein.

In enteric bacteria, the transcription factor σ(E) maintains membrane homeostasis by inducing synthesis of proteins involved in membrane repair and two small regulatory RNAs (sRNAs) that down-regulate synthesis of abundant membrane porins. Here, we describe the discovery of a third σ(E)-dependent sRNA, MicL (mRNA-interfering complementary RNA regulator of Lpp), transcribed from a promoter located within the coding sequence of the cutC gene. MicL is synthesized as a 308-nucleotide (nt) primary transcript that is processed to an 80-nt form. Both forms possess features typical of Hfq-binding sRNAs but surprisingly target only a single mRNA, which encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell. We show 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. This observation raises the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. We also report that σ(E) activity is sensitive to Lpp abundance and that MicL and Lpp comprise a new σ(E) regulatory loop that opposes membrane stress. Together MicA, RybB, and MicL allow σ(E) to repress the synthesis of all abundant outer membrane proteins in response to stress.

Synthetic dual-function RNA reveals features necessary for target regulation.

Small base pairing RNAs (sRNAs) and small proteins comprise two classes of regulators that allow bacterial cells to adapt to a wide variety of growth conditions. A limited number of transcripts encoding both of these activities, regulation of mRNA expression by base pairing and synthesis of a small regulatory protein, have been identified. Given that few have been characterized, little is known about the interplay between the two regulatory functions. To investigate the competition between the two activities, we constructed synthetic dual-function RNAs, hereafter referred to as MgtSR or MgtRS, comprised of the Escherichia coli sRNA MgrR and the open reading frame encoding the small protein MgtS. MgrR is a 98 nt base pairing sRNA that negatively regulates eptB encoding phosphoethanolamine transferase. MgtS is a 31 aa small inner membrane protein that is required for the accumulation of MgtA, a magnesium (Mg2+) importer. Expression of the separate genes encoding MgrR and MgtS is normally induced in response to low Mg2+ by the PhoQP two-component system. By generating various versions of this synthetic dual-function RNA, we probed how the organization of components and the distance between the coding and base pairing sequences 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. IMPORTANCE Dual-function RNAs in bacteria encode a small protein and also base pair with mRNAs to act as small, regulatory RNAs. Given that only a limited number of dual-function RNAs have been characterized, further study of these regulators is needed to increase understanding of their features. This study demonstrates that a functional synthetic dual-regulator can be constructed from separate components and used to study the functional organization of dual-function RNAs, with the goal of exploiting these regulators.

Stem-loops direct precise processing of 3' UTR-derived small RNA MicL.

Increasing numbers of 3'UTR-derived small, regulatory RNAs (sRNAs) are being discovered in bacteria, most generated by cleavage from longer transcripts. The enzyme required for these cleavages has been reported to be RNase E, the major endoribonuclease in enterica bacteria. Previous studies investigating RNase E have come to a range of different conclusions regarding the determinants for RNase E processing. To better understand the sequence and structure determinants for the precise processing of a 3' UTR-derived sRNA, we examined the cleavage of multiple mutant and chimeric derivatives of the 3' UTR-derived MicL sRNA in vivo and in vitro. Our results revealed that tandem stem-loops 3' to the cleavage site define optimal, correctly-positioned cleavage of MicL and probably other sRNAs. Moreover, our assays of MicL, ArcZ and CpxQ showed that sRNAs exhibit differential sensitivity to RNase E, likely a consequence of a hierarchy of sRNA features recognized by the endonuclease.

Dual function of the McaS small RNA in controlling biofilm formation.

Many bacterial small RNAs (sRNAs) regulate gene expression through base-pairing with mRNAs, and it has been assumed that these sRNAs act solely by this one mechanism. Here we report that the multicellular adhesive (McaS) sRNA of Escherichia coli uniquely acts by two different mechanisms: base-pairing and protein titration. Previous work established that McaS base pairs with the mRNAs encoding master transcription regulators of curli and flagella synthesis, respectively, resulting in down-regulation and up-regulation of these important cell surface structures. In this study, we demonstrate that McaS activates synthesis of the exopolysaccharide ß-1,6 N-acetyl-D-glucosamine (PGA) by binding the global RNA-binding protein CsrA, a negative regulator of pgaA translation. The McaS RNA bears at least two CsrA-binding sequences, and inactivation of these sites compromises CsrA binding, PGA regulation, and biofilm formation. Moreover, ectopic McaS expression leads to induction of two additional CsrA-repressed genes encoding diguanylate cyclases. Collectively, our study shows that McaS is a dual-function sRNA with roles in the two major post-transcriptional regulons controlled by the RNA-binding proteins Hfq and CsrA.

The small protein MgtS and small RNA MgrR modulate the PitA phosphate symporter to boost intracellular magnesium levels.

In response to low levels of magnesium (Mg2+ ), the PhoQP two component system induces the transcription of two convergent genes, one encoding a 31-amino acid protein denoted MgtS and the second encoding a small, regulatory RNA (sRNA) denoted MgrR. Previous studies showed that the MgtS protein interacts with and stabilizes the MgtA Mg2+ importer to increase intracellular Mg2+ levels, while the MgrR sRNA base pairs with the eptB mRNA thus affecting lipopolysaccharide modification. Surprisingly, we found overexpression of the MgtS protein also leads to induction of the PhoRB regulon. Studies to understand this activation showed that MgtS forms a complex with a second protein, PitA, a cation-phosphate symporter. Given that the additive effect of ∆mgtA and ∆mgtS mutations on intracellular Mg2+ concentrations seen previously is lost in the ∆pitA mutant, we suggest that MgtS binds to and prevents Mg2+ leakage through PitA under Mg2+ -limiting conditions. Consistent with a detrimental role of PitA in low Mg2+ , we also observe MgrR sRNA repression of PitA synthesis. Thus, PhoQP induces the expression of two convergent small genes in response to Mg2+ limitation whose products act to modulate PitA at different levels to increase intracellular Mg2+ .