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.

Structure of a bacterial OapB protein with its OLE RNA target gives insights into the architecture of the OLE ribonucleoprotein complex.

The OLE (ornate, large, and extremophilic) RNA class is one of the most complex and well-conserved bacterial noncoding RNAs known to exist. This RNA is known to be important for bacterial responses to stress caused by short-chain alcohols, cold, and elevated Mg2+ concentrations. These biological functions have been shown to require the formation of a ribonucleoprotein (RNP) complex including at least two protein partners: OLE-associated protein A (OapA) and OLE-associated protein B (OapB). OapB directly binds OLE RNA with high-affinity and specificity and is believed to assist in assembling the functional OLE RNP complex. To provide the atomic details of OapB-OLE RNA interaction and to potentially reveal previously uncharacterized protein-RNA interfaces, we determined the structure of OapB from Bacillus halodurans alone and in complex with an OLE RNA fragment at resolutions of 1.0 Å and 2.0 Å, respectively. The structure of OapB exhibits a K-shaped overall architecture wherein its conserved KOW motif and additional unique structural elements of OapB form a bipartite RNA-binding surface that docks to the P13 hairpin and P12.2 helix of OLE RNA. These high-resolution structures elucidate the molecular contacts used by OapB to form a stable RNP complex and explain the high conservation of sequences and structural features at the OapB-OLE RNA-binding interface. These findings provide insight into the role of OapB in the assembly and biological function of OLE RNP complex and can guide the exploration of additional possible OLE RNA-binding interactions present in OapB.

Ornate, large, extremophilic (OLE) RNA forms a kink turn necessary for OapC protein recognition and RNA function.

Ornate, large, extremophilic (OLE) RNAs represent a class of noncoding RNAs prevalent in Gram-positive, extremophilic/anaerobic bacterial species. OLE RNAs (∼600 nt), whose precise biochemical functions remain mysterious, form an intricate secondary structure interspersed with regions of highly conserved nucleotides. In the alkali-halophilic bacterium Bacillus halodurans, OLE RNA is a component of a ribonucleoprotein (RNP) complex involving at least two proteins named OapA and OapB, but additional components may exist that could point to functional roles for the RNA. Disruption of the genes for either OLE RNA, OapA, or OapB result in the inability of cells to overcome cold, alcohol, or Mg2+ stresses. In the current study, we used in vivo crosslinking followed by OLE RNA isolation to identify the protein YbxF as a potential additional partner in the OLE RNP complex. Notably, a mutation in the gene for this same protein was also reported to be present in a strain wherein the complex is nonfunctional. The B. halodurans YbxF (herein renamed OapC) is homologous to a bacterial protein earlier demonstrated to bind kink turn (k-turn) RNA structural motifs. In vitro RNA-protein binding assays reveal that OLE RNA forms a previously unrecognized k-turn that serves as the natural binding site for YbxF/OapC. Moreover, B. halodurans cells carrying OLE RNAs with disruptive mutations in the k-turn exhibit phenotypes identical to cells lacking functional OLE RNP complexes. These findings reveal that the YbxF/OapC protein of B. halodurans is important for the formation of a functional OLE RNP complex.

Architectures and complex functions of tandem riboswitches.

Riboswitch architectures that involve the binding of a single ligand to a single RNA aptamer domain result in ordinary dose-response curves that require approximately a 100-fold change in ligand concentration to cover nearly the full dynamic range for gene regulation. However, by using multiple riboswitches or aptamer domains in tandem, these ligand-sensing structures can produce additional, complex gene control outcomes. In the current study, we have computationally searched for tandem riboswitch architectures in bacteria to provide a more complete understanding of the diverse biological and biochemical functions of gene control elements that are made exclusively of RNA. Numerous different arrangements of tandem homologous riboswitch architectures are exploited by bacteria to create more 'digital' gene control devices, which operate over a narrower ligand concentration range. Also, two heterologous riboswitch aptamers are sometimes employed to create two-input Boolean logic gates with various types of genetic outputs. These findings illustrate the sophisticated genetic decisions that can be made by using molecular sensors and switches based only on RNA.

Bacillus halodurans OapB forms a high-affinity complex with the P13 region of the noncoding RNA OLE.

Noncoding RNAs (ncRNAs) longer than 200 nucleotides are rare in bacteria, likely because bacterial genomes are under strong evolutionary pressures to maintain a small genome size. Of the long ncRNAs unique to bacteria, the OLE (ornate, large, extremophilic) RNA class is among the largest and most structurally complex. OLE RNAs form a ribonucleoprotein (RNP) complex by partnering with at least two proteins, OapA and OapB, that directly bind OLE RNA. The biochemical functions of the OLE RNP complex remain unknown, but are required for proper adaptation to certain environmental stresses, such as cold temperatures, short chain alcohols, and high magnesium concentrations. In the current study, we used electrophoretic mobility shift assays to examine the binding of OLE RNA fragments by OapB and found that OapB recognizes a small subregion of OLE RNA, including stem P13, with a dissociation constant (KD ) of ∼700 pm Analyses with mutated RNA constructs, and the application of in vitro selection, revealed that strong binding of OLE RNA by OapB requires a stem containing a precisely located single-nucleotide bulge and a GNRA tetraloop. Although the vast majority of bacteria with the ole gene also have the oapB gene, there are many whose genomes contain oapB but lack ole, suggesting that OapB has other RNA partners in some species that might exhibit similar structural features.

A second RNA-binding protein is essential for ethanol tolerance provided by the bacterial OLE ribonucleoprotein complex.

OLE (ornate, large, extremophilic) RNAs comprise a class of structured noncoding RNAs (ncRNAs) found in many extremophilic bacteria species. OLE RNAs constitute one of the longest and most widespread bacterial ncRNA classes whose major biochemical function remains unknown. In the Gram-positive alkaliphile Bacillus halodurans, OLE RNA is abundant, and localizes to the cell membrane by association with the transmembrane OLE-associated protein called OapA (formerly OAP). These characteristics, along with the well-conserved sequence and structural features of OLE RNAs, suggest that the OLE ribonucleoprotein (RNP) complex performs important biological functions. B. halodurans strains lacking OLE RNA (∆ole) or OapA (∆oapA) are less tolerant of cold (20 °C) and short-chain alcohols (e.g., ethanol). Here, we describe the effects of a mutant OapA (called PM1) that more strongly inhibits growth under cold or ethanol stress compared with strains lacking the oapA gene, even when wild-type OapA is present. This dominant-negative effect of PM1 is reversed by mutations that render OLE RNA nonfunctional. This finding demonstrates that the deleterious PM1 phenotype requires an intact RNP complex, and suggests that the complex has one or more additional undiscovered components. A genetic screen uncovered PM1 phenotype suppressor mutations in the ybzG gene, which codes for a putative RNA-binding protein of unknown biological function. We observe that YbzG protein (also called OapB) selectively binds OLE RNA in vitro, whereas a mutant version of the protein is not observed to bind OLE RNA. Thus, YbzG/OapB is an important component of the functional OLE RNP complex in B. halodurans.

Disruption of the OLE ribonucleoprotein complex causes magnesium toxicity in Bacillus halodurans.

OLE RNAs represent an unusual class of bacterial noncoding RNAs common in Gram-positive anaerobes. The OLE RNA of the alkaliphile Bacillus halodurans is highly expressed and naturally interacts with at least two RNA-binding proteins called OapA and OapB. The phenotypes of the corresponding knockouts include growth inhibition when exposed to ethanol or other short-chain alcohols or when incubated at modestly reduced temperatures (e.g. 20°C). Intriguingly, the OapA 'PM1' mutant, which carries two amino acid changes to a highly conserved region, yields a dominant-negative phenotype that causes more severe growth defects under these same stress conditions. Herein, we report that the PM1 strain also exhibits extreme sensitivity to elevated Mg2+ concentrations, beginning as low as 2 mM. Suppressor mutants predominantly map to genes for aconitate hydratase and isocitrate dehydrogenase, which are expected to alter cellular citrate concentrations. Citrate reduces the severity of the Mg2+ toxicity phenotype, but neither the genomic mutations nor the addition of citrate to the medium overcomes ethanol toxicity or temperature sensitivity. These findings reveal that OLE RNA and its protein partners are involved in biochemical responses under several stress conditions, wherein the unusual sensitivity to Mg2+ can be independently suppressed by specific genomic mutations.

Randomized Trial of Personalized Breast Density and Breast Cancer Risk Notification.

BACKGROUND: Despite widespread implementation of mammographic breast density (MBD) notification laws, the impact of these laws on knowledge of MBD and knowledge of breast cancer risk is limited by the lack of tools to promote informed decision-making in practice. OBJECTIVE: To develop and evaluate whether brief, personalized informational videos following a normal mammogram in addition to a legislatively required letter about MBD result can improve knowledge of MBD and breast cancer risk compared to standard care (i.e., legislatively required letter about MBD included with the mammogram result). DESIGN/PARTICIPANTS: Prospective randomized controlled trial of English-speaking women, age 40-74 years, without prior history of breast cancer, receiving a screening mammogram with a normal or benign finding (intervention group n = 235, control group n = 224). INTERVENTION: brief (3-5 min) video, personalized to a woman's MBD result and breast cancer risk. MAIN MEASURES: Primary outcomes were a woman's knowledge of her MBD and risk of breast cancer. Secondary outcomes included whether a woman reported that she discussed the results of her mammogram with her primary care provider (PCP). KEY RESULTS: Relative to women in the control arm, women in the intervention arm had greater improvement in their knowledge of both their personal MBD (intervention pre/post 39.2%/ 77.5%; control pre/post 36.2%/ 37.5%; odds ratio (OR) 5.34 for change for intervention vs. control, 95% confidence interval (CI) 3.87-7.36; p < 0.001) and risk of breast cancer (intervention pre/post: 66.8%/74.0%; control pre/post 67.9%/ 65.2%; OR 1.42, 95% confidence interval (CI) 1.09-1.84; p = 0.01). Women in the intervention group were more likely than those in the control group to report discussing the results of their mammogram with their PCP (p = 0.05). CONCLUSIONS: Brief, personalized videos following mammography can improve knowledge of MBD and personal risk of breast cancer compared to a legislatively mandated informational letter. Trial Registration Clinicaltrials.gov (NCT02986360).

Challenges of ligand identification for the second wave of orphan riboswitch candidates.

Orphan riboswitch candidates are noncoding RNA motifs whose representatives are believed to function as genetic regulatory elements, but whose target ligands have yet to be identified. The study of certain orphans, particularly classes that have resisted experimental validation for many years, has led to the discovery of important biological pathways and processes once their ligands were identified. Previously, we highlighted details for four of the most common and intriguing orphan riboswitch candidates. This facilitated the validation of riboswitches for the signaling molecules c-di-AMP, ZTP, and ppGpp, the metal ion Mn2+, and the metabolites guanidine and PRPP. Such studies also yield useful linkages between the ligands sensed by the riboswitches and numerous biochemical pathways. In the current report, we describe the known characteristics of 30 distinct classes of orphan riboswitch candidates - some of which have remained unsolved for over a decade. We also discuss the prospects for uncovering novel biological insights via focused studies on these RNAs. Lastly, we make recommendations for experimental objectives along the path to finding ligands for these mysterious RNAs.

Biochemical analysis of hatchet self-cleaving ribozymes.

Hatchet RNAs are members of a novel self-cleaving ribozyme class that was recently discovered by using a bioinformatics search strategy. The consensus sequence and secondary structure of this class includes 13 highly conserved and numerous other modestly conserved nucleotides interspersed among bulges linking four base-paired substructures. A representative hatchet ribozyme from a metagenomic source requires divalent ions such as Mg(2+) to promote RNA strand scission with a maximum rate constant of ∼4 min(-1). As with all other small self-cleaving ribozymes discovered to date, hatchet ribozymes employ a general mechanism for catalysis involving the nucleophilic attack of a ribose 2'-oxygen atom on an adjacent phosphorus center. Kinetic characteristics of the reaction demonstrate that members of this ribozyme class have an essential requirement for divalent metal ions and that they might have a complex active site that employs multiple catalytic strategies to accelerate RNA cleavage by internal phosphoester transfer.

Biochemical analysis of pistol self-cleaving ribozymes.

Pistol RNAs are members of a distinct class of self-cleaving ribozymes that was recently discovered by using a bioinformatics search strategy. Several hundred pistol ribozymes share a consensus sequence including 10 highly conserved nucleotides and many other modestly conserved nucleotides associated with specific secondary structure features, including three base-paired stems and a pseudoknot. A representative pistol ribozyme from the bacterium Lysinibacillus sphaericus was found to promote RNA strand scission with a rate constant of ∼10 min(-1) under physiological Mg(2+) and pH conditions. The reaction proceeds via the nucleophilic attack of a 2'-oxygen atom on the adjacent phosphorus center, and thus adheres to the same general catalytic mechanism of internal phosphoester transfer as found with all other classes of natural self-cleaving ribozymes discovered to date. Analyses of the kinetic characteristics and the metal ion requirements of the cleavage reaction reveal that members of this ribozyme class likely use several catalytic strategies to promote the rapid cleavage of RNA.

New classes of self-cleaving ribozymes revealed by comparative genomics analysis.

Enzymes made of RNA catalyze reactions that are essential for protein synthesis and RNA processing. However, such natural ribozymes are exceedingly rare, as evidenced by the fact that the discovery rate for new classes has dropped to one per decade from about one per year during the 1980s. Indeed, only 11 distinct ribozyme classes have been experimentally validated to date. Recently, we recognized that self-cleaving ribozymes frequently associate with certain types of genes from bacteria. Herein we exploited this association to identify divergent architectures for two previously known ribozyme classes and to discover additional noncoding RNA motifs that are self-cleaving RNA candidates. We identified three new self-cleaving classes, which we named twister sister, pistol and hatchet, from this collection, suggesting that even more ribozymes remain hidden in modern cells.

Evidence That Antibiotics Bind to Human Mitochondrial Ribosomal RNA Has Implications for Aminoglycoside Toxicity.

Aminoglycosides are a well known antibiotic family used to treat bacterial infections in humans and animals, but which can be toxic. By binding to the decoding site of helix44 of the small subunit RNA of the bacterial ribosome, the aminoglycoside antibiotics inhibit protein synthesis, cause misreading, or obstruct peptidyl-tRNA translocation. Although aminoglycosides bind helix69 of the bacterial large subunit RNA as well, little is known about their interaction with the homologous human helix69. To probe the role this binding event plays in toxicity, changes to thermal stability, base stacking, and conformation upon aminoglycoside binding to the human cytoplasmic helix69 were compared with those of the human mitochondrial and Escherichia coli helix69. Surprisingly, binding of gentamicin and kanamycin A to the chemically synthesized terminal hairpins of the human cytoplasmic, human mitochondrial, and E. coli helix69 revealed similar dissociation constants (1.3-1.7 and 4.0-5.4 µM, respectively). In addition, aminoglycoside binding enhanced conformational stability of the human mitochondrial helix69 by increasing base stacking. Proton one-dimensional and two-dimensional NMR suggested significant and specific conformational changes of human mitochondrial and E. coli helix69 upon aminoglycoside binding, as compared with human cytoplasmic helix69. The conformational changes and similar aminoglycoside binding affinities observed for human mitochondrial helix69 and E. coli helix69, as well as the increase in structural stability shown for the former, suggest that this binding event is important to understanding aminoglycoside toxicity.

NMR-based Structural Analysis of Threonylcarbamoyl-AMP Synthase and Its Substrate Interactions.

The hypermodified nucleoside N(6)-threonylcarbamoyladenosine (t(6)A37) is present in many distinct tRNA species and has been found in organisms in all domains of life. This post-transcriptional modification enhances translation fidelity by stabilizing the anticodon/codon interaction in the ribosomal decoding site. The biosynthetic pathway of t(6)A37 is complex and not well understood. In bacteria, the following four proteins have been discovered to be both required and sufficient for t(6)A37 modification: TsaC, TsaD, TsaB, and TsaE. Of these, TsaC and TsaD are members of universally conserved protein families. Although TsaC has been shown to catalyze the formation of L-threonylcarbamoyl-AMP, a key intermediate in the biosynthesis of t(6)A37, the details of the enzymatic mechanism remain unsolved. Therefore, the solution structure of Escherichia coli TsaC was characterized by NMR to further study the interactions with ATP and L-threonine, both substrates of TsaC in the biosynthesis of L-threonylcarbamoyl-AMP. Several conserved amino acids were identified that create a hydrophobic binding pocket for the adenine of ATP. Additionally, two residues were found to interact with L-threonine. Both binding sites are located in a deep cavity at the center of the protein. Models derived from the NMR data and molecular modeling reveal several sites with considerable conformational flexibility in TsaC that may be important for L-threonine recognition, ATP activation, and/or protein/protein interactions. These observations further the understanding of the enzymatic reaction catalyzed by TsaC, a threonylcarbamoyl-AMP synthase, and provide structure-based insight into the mechanism of t(6)A37 biosynthesis.

A novel role of cytosolic protein synthesis inhibition in aminoglycoside ototoxicity.

Ototoxicity is a main dose-limiting factor in the clinical application of aminoglycoside antibiotics. Despite longstanding research efforts, our understanding of the mechanisms underlying aminoglycoside ototoxicity remains limited. Here we report the discovery of a novel stress pathway that contributes to aminoglycoside-induced hair cell degeneration. Modifying the previously developed bioorthogonal noncanonical amino acid tagging method, we used click chemistry to study the role of protein synthesis activity in aminoglycoside-induced hair cell stress. We demonstrate that aminoglycosides inhibit protein synthesis in hair cells and activate a signaling pathway similar to ribotoxic stress response, contributing to hair cell degeneration. The ability of a particular aminoglycoside to inhibit protein synthesis and to activate the c-Jun N-terminal kinase (JNK) pathway correlated well with its ototoxic potential. Finally, we report that a Food and Drug Administration-approved drug known to inhibit ribotoxic stress response also prevents JNK activation and improves hair cell survival, opening up novel strategies to prevent and treat aminoglycoside ototoxicity.

Disruption of the bacterial OLE RNP complex impairs growth on alternative carbon sources.

Ornate, large, extremophilic (OLE) RNAs comprise a class of large noncoding RNAs in bacteria whose members form a membrane-associated ribonucleoprotein (RNP) complex. This complex facilitates cellular adaptation to diverse stresses such as exposure to cold, short-chain alcohols, and elevated Mg2+ concentrations. Here, we report additional phenotypes exhibited by Halalkalibacterium halodurans (formerly called Bacillus halodurans) strains lacking functional OLE RNP complexes. Genetic disruption of the complex causes restricted growth compared to wild-type cells when cultured in minimal media (MM) wherein glucose is replaced with alternative carbon/energy sources. Genetic suppressor selections conducted in glutamate MM yielded isolates that carry mutations in or near genes relevant to Mn2+ homeostasis (ykoY and mntB), phosphate homeostasis (phoR), and putative multidrug resistance (bmrCD). These functional links between OLE RNA, carbon/energy management, and other fundamental processes including protein secretion are consistent with the hypothesis that the OLE RNP complex is a major contributor to cellular adaptation to unfavorable growth conditions.

YrdC exhibits properties expected of a subunit for a tRNA threonylcarbamoyl transferase.

The post-transcriptional nucleoside modifications of tRNA's anticodon domain form the loop structure and dynamics required for effective and accurate recognition of synonymous codons. The N(6)-threonylcarbamoyladenosine modification at position 37 (t(6)A(37)), 3'-adjacent to the anticodon, of many tRNA species in all organisms ensures the accurate recognition of ANN codons by increasing codon affinity, enhancing ribosome binding, and maintaining the reading frame. However, biosynthesis of this complex modification is only partially understood. The synthesis requires ATP, free threonine, a single carbon source for the carbamoyl, and an enzyme yet to be identified. Recently, the universal protein family Sua5/YciO/YrdC was associated with t(6)A(37) biosynthesis. To further investigate the role of YrdC in t(6)A(37) biosynthesis, the interaction of the Escherichia coli YrdC with a heptadecamer anticodon stem and loop of lysine tRNA (ASL(Lys)(UUU)) was examined. YrdC bound the unmodified ASL(Lys)(UUU) with high affinity compared with the t(6)A(37)-modified ASL(Lys)(UUU) (K(d) = 0.27 ± 0.20 µM and 1.36 ± 0.39 µM, respectively). YrdC also demonstrated specificity toward the unmodified versus modified anticodon pentamer UUUUA and toward threonine and ATP. The protein did not significantly alter the ASL architecture, nor was it able to base flip A(37), as determined by NMR, circular dichroism, and fluorescence of 2-aminopuine at position 37. Thus, current data support the hypothesis that YrdC, with many of the properties of a putative threonylcarbamoyl transferase, most likely functions as a component of a heteromultimeric protein complex for t(6)A(37) biosynthesis.

Specific RNA-protein interactions detected with saturation transfer difference NMR.

RNA, at the forefront of biochemical research due to its central role in biology, is recognized by proteins through various mechanisms. Analysis of the RNA-protein interface provides insight into the recognition determinants and function. As such, there is a demand for developing new methods to characterize RNA-protein interactions. Saturation transfer difference (STD) NMR can identify binding ligands for proteins in a rather short period of time, with data acquisitions of just a few hours. Two RNA-protein systems involved in RNA modification were studied using STD NMR. The N (6)-threonylcarbamoyltransferase, YrdC, with nucleoside-specific recognition, was shown to bind the anticodon stem-loop of tRNA(Lys)UUU. The points of contact on the RNA were assigned and a binding interface was identified. STD NMR was also applied to the interaction of the archaeal ribosomal protein, L7Ae, with the box C/D K-turn RNA. The distinctiveness of the two RNA-protein interfaces was evident. Both RNAs exhibited strong STD signals indicative of direct contact with the respective protein, but reflected the nature of recognition. Characterization of nucleic acid recognition determinants traditionally involves cost and time prohibitive methods. This approach offers significant insight into interaction interfaces fairly rapidly, and complements existing structural methods.

The RNA Modification Database, RNAMDB: 2011 update.

Since its inception in 1994, The RNA Modification Database (RNAMDB, http://rna-mdb.cas.albany.edu/RNAmods/) has served as a focal point for information pertaining to naturally occurring RNA modifications. In its current state, the database employs an easy-to-use, searchable interface for obtaining detailed data on the 109 currently known RNA modifications. Each entry provides the chemical structure, common name and symbol, elemental composition and mass, CA registry numbers and index name, phylogenetic source, type of RNA species in which it is found, and references to the first reported structure determination and synthesis. Though newly transferred in its entirety to The RNA Institute, the RNAMDB continues to grow with two notable additions, agmatidine and 8-methyladenosine, appended in the last year. The RNA Modification Database is staying up-to-date with significant improvements being prepared for inclusion within the next year and the following year. The expanded future role of The RNA Modification Database will be to serve as a primary information portal for researchers across the entire spectrum of RNA-related research.

Natural Language Processing to Identify Abnormal Breast, Lung, and Cervical Cancer Screening Test Results from Unstructured Reports to Support Timely Follow-up.

Cancer screening and timely follow-up of abnormal results can reduce mortality. One barrier to follow-up is the failure to identify abnormal results. While EHRs have coded results for certain tests, cancer screening results are often stored in free-text reports, which limit capabilities for automated decision support. As part of the multilevel Follow-up of Cancer Screening (mFOCUS) trial, we developed and implemented a natural language processing (NLP) tool to assist with real-time detection of abnormal cancer screening test results (including mammograms, low-dose chest CT scans, and Pap smears) and identification of gynecological follow-up for higher risk abnormalities (i.e. colposcopy) from free-text reports. We demonstrate the integration and implementation of NLP, within the mFOCUS system, to improve the follow-up of abnormal cancer screening results in a large integrated healthcare system. The NLP pipelines have detected scenarios when guideline-recommended care was not delivered, in part because the provider mis-identified the text-based result reports.