Evidence that OLE RNA is a component of a major stress‐responsive ribonucleoprotein particle in extremophilic bacteria

OLE RNA is a ~600‐nucleotide noncoding RNA present in many Gram‐positive bacteria that thrive mostly in extreme environments, including elevated temperature, salt, and pH conditions. The precise biochemical functions of this highly conserved RNA remain unknown, but it forms a ribonucleoprotein (RNP) complex that localizes to cell membranes. Genetic disruption of the RNA or its essential protein partners causes reduced cell growth under various stress conditions. These phenotypes include sensitivity to short‐chain alcohols, cold intolerance, reduced growth on sub‐optimal carbon sources, and intolerance of even modest concentrations of Mg2+. Thus, many bacterial species appear to employ OLE RNA as a component of an intricate RNP apparatus to monitor fundamental cellular processes and make physiological and metabolic adaptations. Herein we hypothesize that the OLE RNP complex is functionally equivalent to the eukaryotic TOR complexes, which integrate signals from various diverse pathways to coordinate processes central to cell growth, replication, and survival.

OLE RNAs are members of an abundant bacterial noncoding RNA (ncRNA) class that was named for its ornate secondary structure, large size, and its common occurrence in extremophilic organisms (Puerta-Fernandez et al., 2006; Figure 1).Many Gram-positive bacteria that are capable of anaerobic metabolism carry an ole gene (Figure 2), whose ~600-nucleotide transcript accumulates to become one of the most abundant RNAs in cells (Wallace et al., 2012).
The ole gene in each species is almost always located immediately upstream of a coding region for a putative membrane-associated protein (AYT26_RS13995 or BH2780) of unknown function, now called OLE-associated protein A or 'OapA' (Harris et al., 2018;Puerta-Fernandez et al., 2006).We speculated that OLE RNA and OapA would form a ribonucleoprotein (RNP) complex that resides at the cell membrane.Indeed, it was demonstrated (Block et al., 2011) that OLE RNA likely forms a 1:2 RNA-to-protein complex with OapA, where the protein is necessary to localize the RNP complex to the membrane.
Deletion of either the ole gene or the oapA gene (or both) was found to cause strong growth inhibition when cells are cultured under cold (e.g., 20°C) or alcohol (e.g., 5% ethanol) stress conditions (Wallace et al., 2012).However, neither component of this RNP complex is required for robust growth under normal culture conditions.Despite these initial findings, progress on understanding the functions of the complex or its components has been slowed by the lack of clues regarding its association with a distinct, well-known biological or biochemical process.This problem originates from the fact that the first OLE RNA representatives were identified by using a computational search strategy that revealed the existence of a novel ncRNA class, rather than by discovering the RNA as a participant in a well-understood biological process or biochemical pathway.
We have previously proposed (Harris & Breaker, 2018;Puerta-Fernandez et al., 2006) that OLE RNA likely functions as a ribozyme, largely due to its size, structural sophistication, and robust evolutionary conservation.Currently, we have no better hypothesis for the primary biochemical function of the RNA.
Since the discovery of OLE RNA (Puerta-Fernandez et al., 2006), we have sought clues regarding the function(s) of OLE RNA and those of the RNP complex by using diverse experimental strategies and methods from the fields of bioinformatics, genetics, biochemistry, and biophysics (Block et al., 2011;Harris et al., 2018Harris et al., , 2019;;Harris & Breaker, 2018;Lyon et al., 2022;Wallace et al., 2012;Widner et al., 2020;Yang et al., 2021).As discussed herein, OLE RNA has now been linked to a surprising number of biochemical and physiological processes.For example, in addition to the growth defects noted above, disruption of the OLE RNP complex causes cells to become highly sensitized to Mg 2+ concentrations (~2 mM or higher) in growth media that are only modestly above that expected to be maintained inside cells (Harris et al., 2019).Most recently, cells lacking a functional OLE RNP complex were found to exhibit strong growth reduction compared to wild-type cells when cultured using otherwise acceptable alternative carbon/energy sources such as glutamate (Breaker Laboratory, manuscript in preparation).However, it is not apparent to us how these four disparate phenotypes could be caused by a single biochemical deficiency.Thus, no simple, unified theme for the function of the OLE RNP complex has previously emerged.
The salt-and alkali-tolerant species Halalkalibacterium halodurans (previously called Bacillus halodurans, among other names, Joshi F I G U R E 1 Consensus sequence and structural model of OLE RNAs.The high-affinity RNA binding site locations of protein partners have been reported previously for OapA (Block et al., 2011), OapB (Widner et al., 2020;Yang et al., 2021), and OapC (Lyon et al., 2022).The binding site of RpsU (ribosomal protein S21) is tentative (Breaker Laboratory, unpublished observations).The OLE RNA consensus model is adapted from a previous publication (Lyon et al., 2022).

ArgR YqeY
Unmapped , 2022;Takami et al., 2000) has been the primary model organism for studying OLE RNA (Wallace & Breaker, 2011).Although it is known that OLE RNA can be biosynthesized as a separate transcript (Wallace et al., 2012), the ole gene of H. halodurans also appears to be expressed as a long RNA transcript along with 10 flanking open reading frames (ORFs) that includes the oapA gene coding for OapA protein (Puerta-Fernandez et al., 2006) (Figure 3a).Furthermore, the sequential arrangement of ORFs in this gene cluster is surprisingly well-conserved throughout many host genomes (Figure 3b), suggesting these genes might be commonly arranged in an operon.
Co-transcription or clustering of genes in bacterial species is often a strong indicator that they are involved in a related biochemical process (Conway et al., 2014;Okuda et al., 2007;Rocha, 2008).Thus, perhaps the functions of the neighboring genes are linked to the biochemical function of OLE RNA.Unfortunately, even though many of the genes in the ole cluster have well-established functions, there has been no reported explanation for why these genes are often proximally located and arranged in the order commonly observed.
Regardless, the association of OLE RNA with this gene cluster further expands the scope of biological processes necessary to consider when searching for the function of this unusual ncRNA.
Recent genetic and biochemical studies indicate that the OLE RNP complex requires binding by at least two additional proteins called OapB (Harris et al., 2018;Widner et al., 2020;Yang et al., 2021) and OapC (Lyon et al., 2022).Furthermore, the complex seems likely to involve additional protein partners.Remarkably, these recent findings again do not point to a single process or pathway in which the OLE RNP complex participates.Rather, these findings further expand the number of links to diverse cellular processes.Herein, we present various details regarding what is known about OLE RNA, F I G U R E 2 Phylogenetic distribution of OLE RNA.Phylogenetic depiction of a sampling of bacterial species depicting the presence (green) or absence of OLE RNA.Phylogeny and species names are derived from the Genome Taxonomy Database R08-RS214 (Parks et al., 2022).

Clostridium
In the sections below, we describe the diversity of biological processes linked to OLE RNA and note how these functions are evocative of the functions of mTOR complexes in mammals and other eukaryotes (Liu & Sabatini, 2020;Saxton & Sabatini, 2017).The two mTOR complexes called mTOR complex 1 (mTORC1) and mTORC2 sense a wide assortment of biochemical signals and stresses to regulate key processes involved in cellular growth, division, maintenance, and cell death (Deleyto-Seldas & Efeyan, 2021;Sabatini, 2017).Their sensory abilities and regulatory actions allow eukaryotic cells to monitor signals that are of fundamental importance to cell viability, and then act by regulating transcription and translation of genes whose functions are important for the cell to make appropriate adaptations.
Presumably, cells from all three domains of life would benefit from monitoring key nutrients and stresses, integrating this information, and then carrying out actions in a highly coordinated manner to best benefit the cell.However, no molecular devices are known in bacteria that serve a broad, integrative role in stress sensing and adaptation analogous to mTOR complexes.Herein, we describe how the RNP complex formed using OLE RNA might serve this important role in certain species of bacteria.We caution readers to consider many of these connections as tentative, and that our discussions and speculations are not meant to provide full proof of the functional similarity between OLE RNA and the comprehensive list of eukaryotic processes relevant to mTOR complexes.

| H . ha lodu ra ns A S A MODEL ORG ANIS M FOR THE S TUDY OF OLE RNA
OLE RNA genes are often found in obligate-or facultative anaerobic Gram-positive bacterial species that are largely considered F I G U R E 3 Genes neighboring the site of OLE RNA transcription are predicted to code for proteins with diverse functions.(a) The specific ole gene cluster observed in Halalkalibacterium halodurans.Positions −5 to +5 have been observed to be co-transcribed with adjacent genes (Puerta-Fernandez et al., 2006).Boxes are colored according to their associated biological processes as indicated in (b), where the gold box indicates a gene relevant to a specific stress response.(b) Consensus arrangement of genes at positions near ole, where the colored bars represent the proportion of the genes that correspond to the predominant gene listed for each position.Gene functions were assigned based on the most common annotations for the gene sequences in RefSeq (O'Leary et al., 2016)."M 2+ ?" denotes a possible function involving divalent metal ion transport.The figure has been adapted from gene association data published previously (Harris et al., 2018).2).To conduct biochemical and genetic experiments on OLE RNA, a bacterial species was sought that is easily cultured and that could be genetically manipulated.H. halodurans, renamed from B. halodurans, was chosen to serve as a model organism for the study of OLE RNA (Puerta-Fernandez et al., 2006).First, a method for creating genetic transformants and genetic knock-out strains was developed to allow more efficient manipulation of the OLE RNA gene (ole) and other relevant genes (Wallace & Breaker, 2011).The ability to create knock-out strains of the ole and oapA genes permitted the eventual identification of the four disparate phenotypes noted above (Breaker Laboratory, manuscript in preparation; Harris et al., 2019;Wallace et al., 2012).H. halodurans also serves as a good model organism for the study of OLE RNP complexes because its ole gene is flanked by most of the genes found in the consensus ole gene cluster (Figure 3), as further discussed below.

| THE OLE RNA TR AN SCRIP T IS OF TEN MADE FROM AN ECLEC TI C G ENE CLUS TER
In most host species, ole is embedded in an unusual cluster of genes (Figure 3b).RT-PCR analysis with H. halodurans indicates that a series of 10 ORFs are produced by co-transcription with at least some of their neighboring genes (Puerta-Fernandez et al., 2006), although there is evidence that the ole gene residing near the center of this cluster also can be transcribed separately (Ko & Altman, 2007;Wallace et al., 2012).This genomic clustering, and at least occasional co-transcription, strongly suggests that the function of OLE RNA is related to the biological processes performed by the protein products of these neighboring genes.
Clustered gene arrangements in bacteria often indicate their protein products contribute to a single metabolic pathway or physiological process.However, the diverse functions of the proteins encoded by the ole gene cluster (see below) currently defy any simple explanation for this association.
The H. halodurans version of the ole gene cluster has some exceptions from the consensus arrangement (Figure 3a).The nadF gene typically present at position +4 is absent, and thus the argR and recN genes typically at positions +5 and +6 are shifted to the left.In position +6 relative to ole, the H. halodurans cluster carries the spoIVB gene, which codes for a protease involved in activating σ K to initiate the final stage of spore formation (Hoa et al., 2002;Xie et al., 2019).It is also notable that spoIVB is the 12th most common gene located within five ORFs upstream and six ORFs downstream of ole among all species analyzed (Figure 4), strongly suggesting that this gene is somehow relevant to the function of the OLE RNP complex.Regardless, the general similarity in the ole gene cluster between H. halodurans and the consensus cluster among species carrying ole (Figure 3) suggests that experimental analyses of the OLE RNP complex in this species will be reflective of the functions of the complex in many other bacteria that also express OLE RNA.

| G ENE PROXIMIT Y RE VE AL S A PROTEIN PARTNER OF OLE RNA E SS ENTIAL FOR ITS FUN C TION
The gene most frequently associated (by proximity) with ole is oapA (Figure 4), which is located immediately downstream of the ole gene (position +1 of the ole gene cluster) in over 90% of the species that carry them (Figure 3b).Moreover, oapA appears to be present only in cells that also carry a gene for OLE RNA.As noted above, OapA (a ~200 amino acid protein of unknown function) is predicted to form a transmembrane protein and was shown to bind in a 1:2 RNA-toprotein complex with OLE RNA (Block et al., 2011).The resulting RNP complex is localized to the cell's membrane, presumably due to the predicted transmembrane architecture of the protein (Block et al., 2011).
These observations helped formulate the now-proven hypothesis that the products of ole and oapA are components of a natural RNP complex (Harris et al., 2018).Genetic disruption of either ole or oapA cause identical phenotypes involving sensitivity to cold, alcohol, Mg 2+ , and carbon source (Breaker Laboratory, manuscript in preparation; Harris et al., 2018Harris et al., , 2019;;Wallace et al., 2012), demonstrating that the two polymers are essential for the function(s) of the mature form(s) of the OLE RNP complex.In this case, genomic proximity was an accurate indicator of the physical and functional links between OLE RNA and the OapA protein.Further discussions below are prompted by the possibility that the conserved arrangement of other ole-proximal genes likewise indicates that they are somehow relevant to the function of OLE RNP complexes.

| CORRE S P ONDEN CE B E T WEEN THE ole G ENE CLUS TER AND THE B I OLOG I C AL FUN C TIONS OF mTORC1
Despite the disparate functions of the proteins coded by genes in the ole cluster, specific genes are commonly present, and are frequently located in the same position relative to ole.The predominant gene located at each position approaches or exceeds 50% representation (Figure 3b).Furthermore, when a gene is absent in its most common location, it is frequently located immediately adjacent to its most common location.This persistent arrangement of the ole cluster suggests that the locations of these genes are strongly favored through evolution, implying that the functions of the protein products are somehow related to each other and/or to the functions of OLE RNA.
Except for the partnership between OLE RNA and OapA (Block et al., 2011), no other proteins expressed by the gene cluster have been previously proven to directly interact with OLE RNA in a biologically meaningful manner.Although, intriguingly, we have recently identified ArgR (the 7th most common protein encoded by ole gene clusters) (Figure 4) as a possible natural partner for OLE RNA (Lyon et al., 2022).Moreover, we have not been able to identify a simple common theme for how most of the protein products encoded by the ole gene cluster might be functionally associated.In this section, we review the predominant genes neighboring ole and highlight the roles these genes perform in diverse biological and biochemical processes that are evocative of those performed by mTORC1 found in eukaryotes (Liu & Sabatini, 2020;Saxton & Sabatini, 2017).
If the observed gene clustering (Figures 3 and 4) is relevant to the function of OLE RNA, and if the gene annotations appearing in the genomic sequence databases are accurate, then several fundamental biological and biochemical functions can be linked to the OLE RNP complex.These include (i) ribosome biogenesis (nusB, yqxC), (ii) folate-mediated single-carbon management (folD), (iii) DNA repair (xseA, xseB, recN), (iv) isoprenoid biosynthesis (ispA, dxs), (v) NADPmediated hydride (redox) management (nadF), and (vi) arginine/ nitrogen metabolism (argR).We now recognize that these general cellular activities are similar to some of the activities associated with  Sabatini, 2020; Sabatini, 2017; Saxton & Sabatini, 2017).Below, we discuss certain details of these groups of biological activities derived from the gene products from typical ole gene clusters and note how these are related to the known functions of mTORC1.

| Ribosome biogenesis
Both nusB and yqxC commonly found at positions −5 and +3 of the ole cluster, respectively, are annotated as genes coding for proteins involved in rRNA production.NusB proteins (Altieri et al., 2000) have been shown to be important for transcription (Bubunenko et al., 2013;Doherty et al., 2006) and proper folding (Bubunenko et al., 2013;Singh et al., 2016) of bacterial rRNA molecules, whereas YqxC proteins are putative rRNA methyltransferase enzymes that use S-adenosylmethionine to modify the ribose 2′-oxygen atom of a conserved guanosine moiety in the peptidyl transferase site of 23S rRNA F I G U R E 4 Rank order of genes most associated with ole.The 'count', or number of times a gene resides within 5 ORFs upstream or 6 ORFs downstream of ole was established by evaluating the genomes of 730 species that carry OLE RNA.The annotated functions of gene products are grouped into biological processes as indicated by the colored boxes as described in Figure 3.

Comments
OapA (BH2780) forms a 2:1 complex with OLE RNA, localizes to membranes, and OapA (BH2780) forms a 2:1 complex with OLE RNA, localizes to membranes, and possibly functions as a divalent metal cation transporter.possibly functions as a divalent metal cation transporter.Spo0A is the master regulator of sporulation.Spo0A is the master regulator of sporulation.32 32 (Grosjean et al., 2014).These functions presumably are important for assembling the structure of translation-active ribosome particles.
Intriguingly, additional links between the OLE RNP complex and proteins (RpsU and YqeY) relevant to bacterial ribosome function have recently been established, as described in more detail in a later section.
In part, mTORC1 regulates the transcription and processing of rRNAs in eukaryotes via signals generated in response to the levels of oxygen, certain amino acids, or specific signaling molecules (Iadevaia et al., 2014;Liu & Sabatini, 2020).This regulation appears to occur indirectly, wherein the mTOR protein kinase phosphorylates substrates that are involved in ribosome biogenesis (Chauvin et al., 2013), among other functions.

| Single-carbon management
Commonly at position −4 of the ole gene cluster is folD (Shen et al., 1999), which codes for a bifunctional enzyme that produces 10-formyl-tetrahydrofolate (10-formyl-THF).This compound is one of several folate-derived enzyme cofactors that carry single-carbon units with different numbers of bonds between the carbon center and the more electronegative atoms of nitrogen or oxygen.In some bacteria, a deficit of 10-formyl-THF causes an accumulation of the purine biosynthetic intermediate AICAR, which is converted to its triphosphorylated form called ZTP (Bochner & Ames, 1982).Based on riboswitch gene associations, ZTP signals the cell to boost the expression of genes whose protein products catalyze reactions to overcome the 10-formyl-THF deficit or promote certain steps of the purine nucleotide biosynthetic pathway (Kim et al., 2015).Thus, expression of the folD gene is part of the process by which bacterial cells manage the availability of several forms of single carbon units, which is critical for many biochemical pathways including nucleotide biosynthesis.
Single-carbon management via folate metabolism is also regulated by mTORC1, which is the demonstrated mechanism for de novo purine biosynthesis regulation in eukaryotes (Ben-Sahra et al., 2016).Presumably, mTORC1 evaluates various signals to determine whether the cell should commit resources to purine biosynthesis to produce RNA and DNA polymers.Mismanagement of nucleotide biosynthesis has been shown to be mutagenic due to the dedication of limited nucleotides to rRNA biogenesis (Valvezan et al., 2017).Nucleotide levels otherwise can drop to an extent that compromises accurate DNA replication.This provides a rationale for why mTORC1 is involved in managing rRNA biogenesis and nucleotide synthesis, as well as DNA repair as discussed in more detail in the next subsection.

| DNA repair
Three genes common to ole gene clusters, xseA (−3), xseB (−2), and recN (+6), are central participants in DNA repair processes.In Escherichia coli, exonuclease VII is formed by a heteromeric complex involving a single XseA (large subunit) and multiple XseB (small subunit) proteins (Poleszak et al., 2012).The complex is known to degrade single-stranded DNA in the process of DNA damage repair (Vales et al., 1982).Intriguingly, overexpression of XseA in the absence of a concurrent increase in XseB causes a form of apoptotic cell death involving induced DNA damage (Jung et al., 2015), suggesting that the large subunit of endonuclease VII might have roles both in DNA repair and in the introduction of DNA damage during the process of bacterial programmed cell death (Allocati et al., 2015;Bayles, 2014).In addition, the bacterial RecN protein is a cohesin-like protein that interacts with double-stranded DNA to promote DNA ligation (Reyes et al., 2010).Bacterial strains that carry disruptive mutations in RecN are sensitive to DNA-damaging agents, which is consistent with its involvement in the process of DNA repair.
Similarly, the mTOR complexes are known to be extensively involved in indirectly regulating eukaryotic DNA damage responses (Alao et al., 2021;Ma et al., 2018) and apoptosis (Castedo et al., 2002;Feehan & Shantz, 2016;Yang & Klionsky, 2010).The role of mTOR complexes in these two processes might be rationalized in part because the status of various stresses monitored by the complexes, such as nutrient deprivation, physicochemical damage, or other signals can be directly converted into signals that trigger cells to pause their growth or replication, to repair the resulting damage, or to undergo programmed cell death.Bacterial cells, like their eukaryotic counterparts, would similarly benefit from an ability to coordinate DNA damage responses and perhaps programmed cell death with other fundamental processes that are relevant to genes in the ole cluster in a manner analogous to that carried out by the mTOR complexes.

| Isoprenoid biosynthesis
Two genes, ispA (−1) and dxs (+2), common to the ole cluster are involved in the early production stages of isoprenoid (terpenoid) compounds (Heuston et al., 2012).Isoprenoids are long-chain polymers of natural isoprene-like monomers that have diverse roles in cells, including serving as components of membranes (Nickels et al., 2020).
There is evidence that mTOR activity is involved in altering the production of isoprenoids (Zhou et al., 2015) at least in part by affecting the production or degradation of eukaryotic enzymes involved in IPP biosynthesis (Shen et al., 2020).By manipulating the concentrations of the isoprenoid precursors, mTOR presumably affects many of the diverse derivatives that result from the polymerization of IPP and DMAPP (Shen et al., 2020).Among the many isoprenoid compounds are ubiquinone and its various derivatives, which are essential for the proper function of the electron transport chain in species from all domains of life (Nowicka & Kruk, 2010).Intriguingly, both mTOR and the OLE RNP complex have links to energy metabolism and electron transport, as further discussed in the next subsection.

| NADP-mediated hydride management
The nadF (+4) gene codes for NAD kinase, which converts nicotinamide adenine dinucleotide (NAD + ) into its 2′-phosphorylated form NADP + .Thus, NadF is a key enzyme for establishing the ratio of NAD + to NADP + in cells (Kawai & Murata, 2008).In its oxidized form, NADP + is a coenzyme that accepts hydride units during the process of ribose production via the pentose phosphate pathway.This relationship serves as another link, along with folD, between the OLE RNA and nucleotide biosynthesis.In its reduced form, NADPH serves as the main source of hydride units for anabolic reactions and supplies the reducing power to systems that perform antioxidation activities (Agledal et al., 2010).
The production of NADPH is also a major process controlled by mTORC1 activity (Düvel et al., 2010;Hoxhaj et al., 2019).For example, mTORC1 is involved in the regulation of ribose production via the pentose phosphate pathway (Tsouko et al., 2014), which has been noted to link nucleotide biosynthesis to NADPH production for use in anabolic reactions (Saxton & Sabatini, 2017) and for maintaining the redox status of the cell (Lu, 2009).

| Arginine metabolism
The ole gene cluster carries several genes encoding proteins involved in various fundamental cellular processes as noted above, but we had been particularly intrigued by the presence of argR (+5) as a member of this collection of ole-associated genes.Its protein product, the arginine repressor ArgR (Ni et al., 1999;Park et al., 2016), is known to function as an arginine-sensing genetic factor that plays a central role in regulating genes associated with both catabolic and anabolic pathways involving this and certain other amino acids (Park et al., 2016).
For example, abundant arginine triggers the release of repression of genes such as arginine deiminase, which converts arginine to citrulline (Maghnouj et al., 1998).This can eventually lead to the production of glutamate, glutamine, and proline.Indeed, the ArgR repressor protein is known to have more broad effects on metabolism via its diverse functions (Ghochikyan et al., 2002;Lu et al., 2004), and transcriptomics results indicate that its disruption affects numerous fundamental biological processes including amino acid metabolism, nucleotide and coenzyme biosynthesis, and DNA repair (Botas et al., 2018).
Intriguingly, evidence that the ArgR protein of H. halodurans might be a natural partner of the OLE RNP complex was obtained by RNA 'pull-down' experiments, as noted in a later section.
Notably, arginine regulation of mTORC1 function is one of the most distinctive features among its numerous activities (Takahara et al., 2020).For example, proteins called CASTOR1 and CASTOR2 directly bind arginine, which leads to protein complex changes that activate mTORC1 signaling (Chantranupong et al., 2016;Saxton et al., 2016).The purpose of this signaling is not entirely clear but might be serving as a read-out of the abundance of certain amino acids and other compounds (such as polyamines) that are biosynthetically close to arginine, as well as serving to evaluate the abundance of nitrogen in the cell (Mossmann et al., 2018).

| LE SS FREQUENT MEMB ER S OF THE ole G ENE CLUS TER AL SO REL ATE TO mTORC1 FUN C TION
In the previous section, we addressed the functions of the most common gene at each position in the ole cluster.However, insight might be gained by examining additional genes that are frequently found near ole, but that do not predominate at any single position.
We have already noted the presence of the spoIVB gene adjacent to the ole cluster of H. halodurans, which is the 12th most abundant gene near to ole (Figure 4).The SpoIVB protein contributes to the process of sporulation (Hoa et al., 2002;Xie et al., 2019).Similarly, the spo0A gene, which codes for the master regulator of sporulation (Molle et al., 2003), is the 13th most abundant gene in the ole cluster.
These gene associations serve as links between OLE RNA and bacterial cell differentiation, which are notable given the role mTORC1 plays in regulating cell division in eukaryotes (Cuyàs et al., 2014).
The next most abundant genes found in ole clusters are yqhY, tsaD, and dltE, although the abundances of these genes substantially fall relative to other members of the cluster (Figure 4).The YqhY protein (an Asp23 family protein) is implicated in alkaline shock and cell wall stress responses (Müller et al., 2014), and has also been linked to the regulation of fatty acid biosynthesis (Tödter et al., 2017).Alkaline shock response is a known function of mTORC2, wherein high pH is sensed by the complex, and cell survival is promoted by attenuation of apoptosis (Kazyken et al., 2021).The TsaD protein is part of a protein complex that functions as an N 6 -adenosine threonylcarbamoyltransferase enzyme to modify certain tRNAs (Missoury et al., 2018).The DltE enzyme is predicted to be an enzyme with oxidoreductase activity that is involved in teichoic acid modification of lipids for the production of bacterial cell walls (Perego et al., 1995).These proteins presumably affect translation and cell growth/division processes, respectively, and again both processes are regulated by mTOR complexes in eukaryotes.

| THE KNOWN PROTEIN PARTNER S OF OLE RNA S TRENG THEN LINK S TO D IVER S E CELLUL AR FUN C TIONS
Various methods have been used in recent years (Block et al., 2011;Harris et al., 2018;Lyon et al., 2022) to confirm or implicate the relevance of several different proteins to the formation or function of the OLE RNP complex (Figures 1 and 5).Some of these proteins have known or putative functions that further link OLE RNA to surprisingly diverse biological processes, including translation, metabolite sensing, and elemental ion transport.Furthermore, there is no indication that this collection of proteins represents a complete set of functional affiliates of the OLE RNP complex.Thus, we anticipate that additional cellular processes will be linked to OLE RNA by the discovery of more partners.Below, we discuss each of the confirmed protein partners and, when possible, draw links to the function of mTOR complexes.

| OapA
The oapA gene represents the most common ORF residing adjacent to OLE RNA (Figure 4).In addition, we have established the basic characteristics of the physical association between OapA proteins and the RNA (Block et al., 2011), as described in an earlier section.OapA is essential for OLE RNA localization to membranes and for the general function of the OLE RNP complex (Breaker Laboratory, manuscript in preparation; Block et al., 2011;Harris et al., 2018;Wallace et al., 2012) (Figure 5a,b).Unfortunately, OapA proteins do not exhibit strong homology to other proteins of known function, and therefore its biochemical activity (if any) beyond that described above remains uncertain.However, we speculate here on the possible functions of OapA beyond its proven ability to bind OLE RNA.
The majority of each OapA polypeptide appears to form a cyclin M domain (also called DUF21) (Mistry et al., 2021), which F I G U R E 5 Additional proteins and biological processes linked to the function of the OLE RNP complex.(a) Genes relevant to OLE RNA whose alteration has been examined to determine if cells exhibit stress phenotypes like those observed when the gene for OLE RNA is deleted (Δole).Data is derived from previous studies on OLE RNA."C source" indicates the carbon and energy source present in cultures.(b) Summary of the suppressive effects of the alteration of genes or growth conditions on OLE-related phenotypes observed with Halalkalibacterium halodurans cells.Mn 2+ RS is a Mn 2+ -sensing riboswitch with a deletion disrupting a terminator stem.Note that some mutations suppress the mild phenotypes exhibited by Δole cells, whereas others suppress the severe forms of the phenotypes exhibited by PM1 cells.(c) Overview of the biological or biochemical processes relevant to the proteins or media conditions whose alterations affect OLE RNA phenotypes.M 2+ indicates divalent magnesium or manganese homeostasis.Information regarding the ascribed roles of genes and their links to the OLE RNP complex was derived from various studies on OLE RNA.

Genes
ole/PM1 Phenotype Suppresses Alteration corresponds to the transmembrane region of CNNM (also called ancient conserved domain protein or ACDP) family proteins (Giménez-Mascarell et al., 2019).Many CNNM family proteins from all three domains of life are evolutionarily conserved Mg 2+ transporters that contain a CNNM domain followed by a pair of cystathionineβ-synthase (CBS) domains and often additional variable domains in the cytoplasmic region (Funato & Miki, 2019;Giménez-Mascarell et al., 2019).Notably, OapA diverges from other CNNM family proteins because OapA lacks additional conserved domains that commonly accompany the transmembrane portion of Mg 2+ transporters (Figure 6).OapA also appears to lack the conserved residues in CNNM proteins required for coordinating the Mg 2+ ligand (Chen et al., 2021;Huang et al., 2021).Regardless, this weak sequence similarity to known transporters for Mg 2+ (and citrate) or Mn 2+ from bacterial and eukaryotic species (Harris et al., 2019), is made more noteworthy by the observation that strains lacking a functional OapA protein or OLE RNP complex are strongly sensitive to elevated concentrations of Mg 2+ in growth media (Harris et al., 2019).Given these observations, we speculate that OapA might have a direct role in divalent cation homeostasis (Figure 5c).
Intriguingly, it has been proposed that Mg 2+ is a major regulator of mTOR activity (De Baaij et al., 2015;Rubin, 2005), and evidence for this hypothesis is accumulating (Feeney et al., 2016;Funato et al., 2014;Hardy et al., 2019;Liu et al., 2021;Shindo et al., 2020;Sponder et al., 2018;Yamanaka et al., 2018).Perhaps the regulation of Mg 2+ and/or Mn 2+ levels provide a simple mechanism by which major cellular functions related to cell growth and replication can be controlled (Rubin, 2005).If true, then divalent ion management by the OLE RNP complex might permit cells to integrate nutrient and stress sensing with general mechanisms for cell regulation.If the cation relevant to OapA is Mn 2+ , perhaps its homeostasis is important for the metabolism of certain carbon sources (Papp- Wallace & Maguire, 2006) or for the function of enzymes that defend against oxidative DNA damage (Yesilkaya et al., 2000).The possible relevance of Mn 2+ is notable given that there are links to carbon metabolism and DNA repair in the ole gene cluster as described earlier.
Furthermore, we have recently established that Mn 2+ addition to culture media can overcome two of the four known phenotypes related to OLE RNA: Mg 2+ toxicity and carbon source growth suppression (Breaker Laboratory, manuscript in preparation).
First, both proteins were identified in a genetic selection (Harris et al., 2018) that exploited the fact that a unique OapA protein mutation (PM1 strain) causes all four known growth-restriction phenotypes to become more severe (Harris et al., 2018).If cells acquire disruptive mutations in components of the OLE RNP complex that are essential for its formation or function, then these strains should suppress the severe PM1 growth limitations and exhibit growth characteristics like the Δole or ΔoapA strains.Indeed, OapB and OapC proteins have been proven to be essential components of the OLE RNP complex (Harris et al., 2018;Lyon et al., 2022;Widner et al., 2020).
In addition, both proteins were identified in "pull-down" experiments, wherein binding partners are crosslinked to OLE RNA, the complexes separated from the cellular milieu, and the proteins identified by mass spectrometry (Lyon et al., 2022).Unfortunately, for both OapB and OapC, there are no published links to established biological processes that can provide clues regarding the functions of the OLE RNP complex.Perhaps these two small proteins function only to stabilize substructures of the OLE RNA, thereby assist in the folding of the RNA as it forms its active conformations.
Regardless, these now-proven methods for identifying key components of the OLE RNP complex also revealed numerous other protein candidates, which await careful genetic and biochemical validation studies.One of the most intriguing candidates identified in the pull-down experiments is ArgR, which is notable because the gene for this protein is frequently located in the ole gene cluster, as discussed in detail above.Other candidates include YqeY and RpsU, which are discussed in the next subsection.

| YqeY and RpsU
The proteins YqeY (also called BH1355 or AYT26_RS07050) and RpsU (BH1354, AYT26_RS07045, ribosomal protein S21) were both identified as strong candidate protein partners for OLE RNA in pulldown experiments (Lyon et al., 2022).Notably, YqeY (considered a protein of unknown function) is often encoded in a two-gene operon together with RpsU (Takada et al., 2014).The H. halodurans homolog of YqeY has structural (and modest sequence) similarity to the C-terminal domain of the glutaminyl-tRNA synthetase (GlnRS) of Deinococcus radiodurans, which acts as an affinity enhancer for tRNA Gln (Deniziak et al., 2007).Therefore, a role for YqeY in translation via tRNA binding is anticipated.
RpsU is part of the small (30S) ribosomal subunit and is required for efficient translation initiation, most likely by favoring binding of the 16S rRNA to its Shine-Dalgarno (SD) sequence in mRNAs (Backendorf et al., 1981;Held et al., 1974).In vitro experiments with synthetic RNAs or natural mRNAs demonstrated that ribosomes can F I G U R E 6 OapA proteins carry CNNM domains like divalent metal ion transporters but lack cystathionineβ-synthase (CBS) domains.Numbers identify the range of amino acids that form the CNNM domain for each protein depicted.CorB is a Mg 2+ transporter from the archaeal species Methanoculleus thermophilus (Chen et al., 2021).MpfA is a Mg 2+ transporter from the bacterial species Staphylococcus aureus (Armitano et al., 2016).CNNM4 is a Mg 2+ transporter from human (Giménez-Mascarell et al., 2019).Domain homology was determined using MOTIF Search (genome.jp/tools/motif).only translate these targets either when RpsU is present in the 30S subunit or when secondary structures preceding the start codon are removed by RNase treatment (Backendorf et al., 1981;Van Duin & Wijnands, 1981).Bacterial species that favor the expression of mRNAs lacking 5′-untranslated region (5′-UTR) sequences (leaderless mRNAs or lmRNAs) tend to lack RpsU (Beck & Moll, 2018), suggesting that this protein is required for efficient translation of mRNAs carrying such leaders.

S. aureus
Both YqeY and RpsU proteins have been observed to selectively bind full-length OLE RNA and certain fragments in vitro (Breaker Laboratory, unpublished observations).Given that RpsU is one of the last proteins to assemble on functional ribosomes (Jham et al., 2021)

| G ENE TI C SCREEN S AND OLE RNA PULL-DOWN A SSAYS LINK THE OLE RNP COMPLE X TO ADDITIONAL B IOLOG IC AL PRO CE SS E S
As described above, the OapA PM1 strain (Harris et al., 2018) exhibits more severe versions of the cell growth restriction phenotypes observed in strains wherein OLE RNA, OapA, or both components are genetically disrupted (Breaker Laboratory, manuscript in preparation; Harris et al., 2018Harris et al., , 2019;;Lyon et al., 2022).Thus, genetic suppressor selections can be conducted to identify disruptive mutations in genes whose products are required for the formation of a functional OLE RNP complex or are required for processes associated with the complex.For example, any mutations that disable the OLE RNP complex will cause the severe PM1 phenotypes to convert to the less severe forms observed when the RNP complex is not able to form.
Genetic suppressor selections conducted under three of the four stresses known to cause growth limitations (cold, Mg 2+ , and carbon source stresses) (Breaker Laboratory, manuscript in preparation; Harris et al., 2018Harris et al., , 2019) ) have revealed mutations in genes relevant to a surprising diversity of pathways and processes.Rather than revealing a single pathway relevant to OLE RNA function, these findings greatly expand the number of biochemical pathways and biological processes linked to OLE RNA (Figure 7).Furthermore, some suppressor mutations do not have equivalent effects on all the phenotypes caused by disruption of the RNP complex.These differences indicate that there are likely to be numerous additional cellular components functionally associated with OLE RNA (Figure 7a) that are involved in linking these disparate processes.Some of the most prominent candidates that might have a direct or indirect association with the OLE RNP complex are briefly discussed below.
7.1 | Genetic suppressors reside in RNA and protein targets related to Mn 2+ We have isolated (Breaker Laboratory, manuscript in preparation) a series of robust suppressor strains that overcome the restricted growth of H. halodurans cells lacking a functional OLE RNP complex when cultured in media containing only glutamate as a carbon/ energy source.Intriguingly, Mn 2+ homeostasis is the predominant process affected by the acquired mutations (Figure 7b).Specifically, mutations in a representative of the Mn 2+ riboswitch class (Dambach et al., 2015;Price et al., 2015) or immediately downstream in the coding region for the YkoY protein, previously linked to excess Mn 2+ tolerance (Paruthiyil et al., 2020), are observed.Furthermore, two subunits (MntB and MntC) of a multidomain Mn 2+ transporter (Que & Helmann, 2000) were also observed to be mutated in some suppressor mutant strains.Based on the characteristics of these mutations, we speculated that cells lacking a functional OLE RNP complex were overcoming the growth limitation by increasing their cellular concentration of Mn 2+ .Indeed, supplementation of media with Mn 2+ promotes cell growth under conditions that otherwise cause the carbon source and Mg 2+ stress phenotypes, but this treatment does not overcome the cold-or alcohol-triggered growth defects (Breaker Laboratory, manuscript in preparation).
Some evidence exists that indicates Mn 2+ affects TORC1 function (Devasahayam et al., 2006(Devasahayam et al., , 2007;;Nicastro et al., 2022), and that this mechanism appears to be conserved in mammals (Nicastro et al., 2022).Specifically, it is hypothesized that Mn 2+ levels are associated with the regulation of key cellular processes via mTORC1, including glutamine metabolism (Nicastro et al., 2022).Given that the strongest carbon source stress identified in our study for OLE RNP disruption strains is caused by glutamate, and that this stress can be overcome by Mn 2+ , perhaps cellular Mn 2+ concentrations are balanced with various other biological processes through the OLE RNP complex in bacteria or through the action of TORC1 in eukaryotes.

| Other diverse genetic suppressors
Many additional genetic suppressor selection hits have been identified (Figure 7b), but these are both too numerous and too diverse in annotated functions to cover in detail here.Perhaps the most remarkable suppressor mutations reside in two genes called bmrC and bmrD, which were obtained from selections under both carbon source and Mg 2+ stress conditions (Breaker Laboratory, manuscript in preparation; Harris et al., 2018).Their protein products form a heterodimer (Lin et al., 2005) that has been reported to function as a multidrug efflux pump (Lin et al., 2005) Schematic representation of the OLE RNP complex and proteins known to associate with the particle either as confirmed (Block et al., 2011;Harris et al., 2018;Lyon et al., 2022;Widner et al., 2020;Yang et al., 2021), biologically relevant components (blue) or as proteins known to associate with the particle but where the biological relevance of the interaction is uncertain (orange).For example, pull-down (Lyon et al., 2022) and RNA binding assays reveal OLE binding by BH3508, which is annotated as a DNA cytidine methyltransferase and is part of a two-gene restriction-modification system.The (x) notations indicate that multiple copies of the protein appear to be bound by OLE RNA in vitro.(b) Candidate proteins relevant to the formation and or function of the OLE RNP complex.Candidates were identified via the acquisition of mutations resulting from suppressor selection studies (Breaker Laboratory, manuscript in preparation; Harris et al., 2018Harris et al., , 2019)).Proteins that have been experimentally proven to serve as biologically relevant components of the OLE RNP complex (blue) or that have been shown to bind OLE RNA (orange) are designated.Asterisks identify proteins that appear in more than one data set derived from genetic suppressor selections, RNA pull-down assays, or the ole gene cluster.(c) Candidate proteins for direct binding to OLE RNA as identified by OLE RNA pull-down experiments (Lyon et al., 2022).
the suppressor selection in the carbon-source phenotype suggests that its effects on cells might be broader than just overcoming the Mg 2+ stress phenotype.

| OLE RNA pull-downs expand the list of protein partner candidates
Another productive means to identify components of the OLE RNP complex is an RNA pull-down method called CHART (Simon, 2013).
Specifically, biotinylated DNA oligonucleotides are used to hybridize to OLE RNA derived from extracts of cells wherein RNAprotein complexes have been chemically crosslinked.By a process of streptavidin-mediated affinity capture, selective degradation of RNA-DNA hybrids, reversal of crosslinks, and protein mass spectrometry analysis, the identities of possible protein partners in the complex are obtained.This approach has identified both OapB and OapC (Figure 7c) as high-ranking candidates.These two proteins were also prominent hits in certain suppressor selections (Figure 7b) and had previously been experimentally validated as essential components of the OLE RNP complex (Harris et al., 2018;Lyon et al., 2022).Intriguingly, the list of strong candidates identified from CHART assays is long (Lyon et al., 2022) (Figure 7c), and the total list that also includes weak candidates (not shown) is much longer.Although the remaining candidates await validation studies, it is likely that OLE RNA interacts with additional protein partners.
If this is true, then the RNA has even deeper connections to diverse biological processes than has been demonstrated to date.

| S PECUL ATI ON ON THE FUN C TI ON OF OLE RNA
The conserved nucleotide sequences and extensive structural features suggest to us that OLE RNA has one or more biochemical functions that go beyond just serving as a scaffold for binding proteins.
Other bacterial structured ncRNAs that are large, highly conserved, and widely distributed usually function as ribozymes (Harris & Breaker, 2018).Therefore, a ribozyme function that somehow contributed to regulating cell growth and stress responses remains our lead hypothesis.Perhaps the various functions of proteins physically or functionally linked to OLE RNA can be evaluated to derive clues regarding other biochemical functions.
Definitive conclusions regarding the biochemical function of OLE RNA cannot be made at this time, but some intriguing insights are emerging.For example, OapA most closely approximates a CNNM protein without the CBS domains, which inspires our hypothesis that OLE RNAs might perform roles like those served by these missing protein domains.For example, various CBS domains are known to bind ligands with adenosyl moieties (Baykov et al., 2001;Scott et al., 2004), such as ATP (Li et al., 2015;Tseng et al., 2011) or c-di-AMP (Heidemann et al., 2022;Huynh et al., 2016;Schuster et al., 2016), sometimes to regulate the function of adjoining domains.An intriguing possibility is that OLE RNA functions as a c-di-AMP synthase to manipulate diverse processes (including stress responses) known to be regulated by this nucleotide-derived signaling molecule (Fahmi et al., 2017;Stülke & Krüger, 2020;Zarrella & Bai, 2021).

| CON CLUDING REMARK S
The study of OLE RNA continues to pose substantial challenges primarily because its function does not appear to be limited to a single prominent biological process.Rather, OLE RNA has been linked to a remarkably diverse collection of processes that are fundamental to cell homeostasis, stress responses, growth, replication, and cellular repair.All cells must carry out these fundamental processes and presumably coordinate actions in response to various internal and external stresses.In eukaryotes, the sophisticated sensory roles and genetic/biochemical actions that conditions necessitate are coordinated by TOR complexes (Liu & Sabatini, 2020;Sabatini, 2017;Saxton & Sabatini, 2017).However, no multicomponent device has been previously identified that serves such a diversity of roles in bacteria.
We propose that OLE RNA is a major component of an RNP complex that performs at least some of the essential roles in certain bacteria that are served by the mTOR complexes in mammals and in other eukaryotes by similar complexes.Perhaps the OLE RNP complex senses stresses by their effects on the structure of the OLE RNA or its protein partners.Similarly, adaptations to these stresses might be mediated by the RNA, its protein partners, or by one or more signaling molecules whose concentrations are manipulated by this unusual particle.
In recognition of this potential functionally analogous system, we propose naming the OLE RNP particle the "bacterial Tasks OLE Regulates" complex, or the "bTOR" complex.We do not intend to suggest the components of the OLE RNP complex are evolutionary homologs of the components of the mTOR complexes.Rather, the complexes in these different domains of life have likely evolved similar functions through convergent evolution because stresses and biochemical responses are similar for cells regardless of their classification into different domains of life.Likewise, although we cannot be certain that the broader biological tasks performed by the OLE RNP complex are perfectly superimposable on the functions carried out by mTOR complexes, it seems certain that additional major functions will be linked to this bacterial system as further studies are pursued.
There also remain major questions regarding how bacteria that lack OLE RNA address the challenges of coordinating fundamental biological processes based on nutrient and stress inputs.Many Gram-positive species express OLE RNA and OapA from the central region of the ole gene cluster, but some species lack the genes for these two biopolymers while retaining a near-identical cluster of genes.Even in evolutionarily distant bacterial species, some of the genes in the cluster remain proximal in the genome.These arrangements again suggest that the protein products of genes in the cluster remain functionally related despite the absence of OLE RNA.Perhaps the functions of these varied proteins in organisms that lack OLE RNA are coordinated either by direct physical contact or by signaling processes yet to be fully elucidated.
partners, and its possible associations with other proteins.Based on the known or predicted roles of the components in the complex, we speculate that the OLE RNP complex functions to sense numerous cellular stresses and responds by potentially affecting a broad set of fundamental biological processes important for maintaining cell growth, nutrient homeostasis, cell integrity, and DNA damage repair, among other key physiological and metabolic adaptations.This list of putative functions resembles those performed by the mTOR complexes essential for guiding key processes eukaryotic mTOR complexes (Deleyto-Seldas & Efeyan, 2021; Liu &