Recombinant Oligoribonuclease (orn)

What is the primary function of Oligoribonuclease (ORN) in bacterial cells?

Oligoribonuclease (ORN) functions as a 3'-5' exoribonuclease that specifically degrades small RNA oligomers, particularly those comprising two to five nucleotides (commonly referred to as nanoRNAs) . ORN performs the critical final step in RNA decay pathways, converting oligoribonucleotides to monoribonucleotides . This function is essential for cell viability in Escherichia coli, as the accumulation of short oligoribonucleotides can be deleterious to cellular processes .

Methodologically, researchers have demonstrated ORN's function through knockout studies and complementation experiments. In E. coli, cellular viability depends on either RNase T or PNPase functioning in conjunction with ORN, highlighting potential backup mechanisms for this essential activity .

What types of RNA substrates does ORN preferentially degrade?

ORN exhibits strong preference for RNA oligomers consisting of 2-5 nucleotides, known as nanoRNAs . In Pseudomonas aeruginosa, recombinant ORN has demonstrated particular activity toward 5'-phosphoguanylyl-(3',5')-guanosine (pGpG), a dinucleotide resulting from the hydrolysis of cyclic diguanylate (c-di-GMP) by EAL-domain phosphodiesterases .

Substrate preference can be experimentally determined through:

• In vitro degradation assays using synthetic RNA oligomers

• Analysis of accumulated small RNA species in ORN-deficient strains

• Comparative kinetic studies with various substrate lengths

This specificity for very short RNA fragments distinguishes ORN from other exoribonucleases that typically process longer RNA molecules, positioning it at the final stage of RNA degradation pathways.

How conserved is ORN across different bacterial species?

ORN is highly conserved among various bacterial taxa, particularly "among Actinobacteria, Beta-, Delta- and Gammaproteobacteria" . This strong conservation indicates ORN performs a fundamental function in bacterial RNA metabolism that has been maintained throughout evolutionary history.

The enzyme has been specifically studied in:

• Escherichia coli (where it is essential for viability)

• Pseudomonas aeruginosa (where it regulates c-di-GMP signaling)

• Bacillus subtilis (where YtqI functions as an ORN analog)

• Caulobacter crescentus (whose ORN structure has been determined)

Comparative genomic approaches reveal ORN orthologs share high sequence similarity in catalytic domains across these diverse bacterial species, suggesting strong selective pressure to maintain this enzymatic function.

What is the structural organization of the ORN enzyme?

The structural organization of ORN involves multimeric complexes, though the exact quaternary structure may vary by organism. Evidence from studies on the human homolog REXO2 demonstrates that the enzyme forms a homotetramer. When analyzed by Blue Native PAGE, mitochondrial REXO2 appeared as a single 90-100 kDa complex, consistent with a tetrameric arrangement of four mature monomers (each approximately 24.4 kDa) .

The structural data available for ORN includes:

The crystal structure of Caulobacter crescentus ORN (CpsORN) was solved using molecular replacement with the Haemophilus influenzae ortholog (55% sequence identity) as template . The structure provides insights into the enzyme's catalytic mechanism and substrate binding.

What are the known homologs of bacterial ORN in eukaryotic cells?

The primary known eukaryotic homolog of bacterial ORN is REXO2 in humans and Saccharomyces cerevisiae (where it's also known as YNT20 or REX2p) . These homologs share the core function of degrading small RNA oligomers, though with some notable differences in localization and potentially broader physiological roles.

Key features of REXO2 include:

• Dual cellular localization in humans:

  • Present in both cytosolic and mitochondrial fractions

  • Within mitochondria, found in both the intermembrane space and matrix

• Functions in yeast (YNT20/REX2p):

  • Initially characterized as exclusively mitochondrial

  • Functions in the mitochondrial DNA escape pathway mediated by YME1

  • Required for processing various nuclear RNAs, including U4, U5L, U5S snRNAs and components of RNase P and 5.8S rRNA

The dual localization of REXO2 suggests that this enzyme plays important roles in RNA metabolism in multiple cellular compartments, potentially coordinating RNA processing activities between these compartments.

How does ORN contribute to cyclic diguanylate (c-di-GMP) signaling in bacteria?

ORN plays a critical role in cyclic diguanylate (c-di-GMP) signaling by degrading 5'-phosphoguanylyl-(3',5')-guanosine (pGpG), the intermediate product of c-di-GMP degradation . The c-di-GMP pathway controls diverse cellular processes in bacteria, with c-di-GMP being synthesized by diguanylate cyclases and degraded by phosphodiesterases (PDEs).

The mechanistic pathway involves:

• EAL-domain PDEs hydrolyze one phosphodiester bond in c-di-GMP, producing pGpG

• ORN then degrades pGpG to prevent its accumulation

• In ORN-deficient cells (Δorn mutant), pGpG accumulates

• Elevated pGpG exerts product inhibition on EAL-dependent PDEs (PA2133, PvrR, and RocR)

• This inhibition increases intracellular c-di-GMP levels

• Higher c-di-GMP leads to overexpression of extracellular polymers and biofilm production

Experimentally, researchers demonstrated that adding ORN alleviates pGpG-dependent inhibition of PDEs, confirming that "ORN provides homeostatic control of intracellular pGpG under native physiological conditions" . This regulatory mechanism represents a fundamental aspect of c-di-GMP signal transduction in bacteria.

What methodological approaches can detect changes in ORN activity in vivo?

Several methodological approaches can effectively detect changes in ORN activity in bacterial cells:

• Quantification of nanoRNA accumulation:

  • Extract small RNAs from wild-type and ORN-depleted cells

  • Use high-resolution gel electrophoresis or mass spectrometry to detect 2-5 nucleotide species

  • Compare profiles to identify ORN-dependent changes

• pGpG accumulation assay:

  • Measure pGpG levels using liquid chromatography-mass spectrometry (LC-MS)

  • Elevated pGpG levels indicate reduced ORN activity

• Phenotypic assessment:

  • Monitor biofilm formation (increases with reduced ORN activity)

  • Assess extracellular polymer production

  • Measure c-di-GMP-dependent behaviors (motility, adhesion)

• Genetic complementation:

  • Express recombinant ORN in ORN-deficient strains

  • Measure restoration of normal phenotypes and RNA profiles

  • Use expression vectors with tunable promoters to establish dose-response relationships

• Interaction studies:

  • Co-immunoprecipitation to identify protein partners

  • Bacterial two-hybrid systems to detect regulatory interactions

  • RNA-binding assays to characterize substrate recognition

These approaches can be combined to comprehensively assess ORN activity under various experimental conditions, providing insights into the enzyme's regulation and physiological roles.

How do cells compensate for ORN deficiency in experimental models?

Research indicates that cells possess backup mechanisms to compensate for ORN deficiency. In E. coli, the absence of ORN alone does not cause immediate cessation of growth; rather, cellular viability becomes dependent on either RNase T or PNPase functioning in conjunction with ORN . This finding identifies both RNase T and PNPase as potential compensatory mechanisms for oligoribonuclease activity.

The experimental approach to studying these compensatory mechanisms includes:

• Construction of conditional mutants:

  • E. coli orn mutant (strain UM341) using anhydrotetracycline (Atc)-inducible promoter PLtetO-1 with Tet-repressor (TetR)

  • Allows controlled depletion of ORN while avoiding lethal effects

• Double knockout studies:

  • Creating orn/rnaseT and orn/pnpase double mutants

  • Assessing synthetic lethality phenotypes

  • Determining which combinations are viable under various conditions

• Transcriptomic and proteomic analysis:

  • Identifying upregulated exoribonucleases in ORN-deficient strains

  • Characterizing changes in expression of RNA processing machinery

These approaches reveal that bacterial cells have evolved redundant mechanisms to ensure the essential function of degrading small RNA oligomers can continue even when the primary enzyme (ORN) is compromised.

What is the mechanistic basis for ORN's preference for small RNA oligomers?

The mechanistic basis for ORN's preference for small RNA oligomers relates to its structural features and catalytic properties. The crystal structure of ORN from Caulobacter crescentus (CpsORN) provides insights into this specificity .

Key structural determinants likely include:

• Active site architecture:

  • Designed to accommodate very short RNA molecules (2-5 nucleotides)

  • Constrains longer substrates from proper positioning

• Substrate binding pocket:

  • Specific interactions with terminal nucleotides

  • Limited space that excludes longer oligonucleotides

• Catalytic mechanism:

  • 3'→5' exonucleolytic activity

  • Hydrolysis of phosphodiester bonds in RNA

The enzyme's preference for nanoRNAs aligns with its biological role in the final stages of RNA degradation pathways, where it completes the process by degrading the smallest RNA fragments that other exoribonucleases cannot efficiently process.

Analysis of ORN bound to substrate analogs like pNP-TMP has provided additional insights into the enzyme's catalytic mechanism and substrate recognition , though further structural and biochemical studies are needed to fully elucidate the molecular determinants of substrate specificity.

How is ORN expression regulated in bacterial cells?

While the search results provide limited information about ORN expression regulation, several insights can be gleaned:

• Promoter identification:

  • Researchers have "determined the true transcription start site of orn, and have identified the putative promoter sequence, contrary to what is currently annotated"

  • This finding suggests "orn regulation is more intricate than previously thought"

• Experimental approaches to study regulation:

  • 5' RACE (Rapid Amplification of cDNA Ends) to map transcription start sites

  • Reporter gene fusions to characterize promoter activity

  • ChIP-seq to identify transcription factor binding sites

• Construction of regulated expression systems:

  • Anhydrotetracycline (Atc)-inducible promoter systems have been used to control ORN expression

  • These systems allow for tunable expression levels to study dosage effects

Understanding ORN regulation is particularly important given its essential nature in many bacteria. Further research into transcriptional, post-transcriptional, and post-translational regulation mechanisms would provide valuable insights into how bacteria maintain appropriate levels of this critical enzyme under different environmental conditions.

What are the optimal conditions for expressing and purifying recombinant ORN?

While the search results don't provide a detailed protocol, several methodological considerations for expressing and purifying recombinant ORN can be inferred:

• Expression system selection:

  • E. coli BL21(DE3) or similar strains optimized for recombinant protein expression

  • Consider codon optimization for the specific ORN ortholog being produced

  • Inducible promoter systems (T7, tac) for controlled expression

• Protein tagging strategies:

  • N-terminal or C-terminal His-tag for affinity purification

  • Consider tag removal options if needed for activity studies

  • GST or MBP fusion for improved solubility if expression yields are low

• Purification scheme:

  • Initial capture: Ni-NTA affinity chromatography for His-tagged proteins

  • Secondary purification: Size exclusion chromatography to isolate tetrameric form

  • Optional: Ion exchange chromatography for removal of nucleic acid contamination

• Buffer optimization:

  • pH range: Typically 7.0-8.0 for optimal stability

  • Salt concentration: 100-300mM NaCl to maintain solubility

  • Presence of reducing agents (DTT, β-mercaptoethanol) to maintain cysteine residues

  • Consider adding glycerol (5-10%) for storage stability

• Activity preservation:

  • Avoid repeated freeze-thaw cycles

  • Store in small aliquots at -80°C for long-term storage

  • Test activity after purification with defined oligoribonucleotide substrates

These parameters should be empirically optimized for each specific ORN ortholog and experimental purpose.

What methods are used to quantitatively assess ORN activity in vitro?

Several methods can be employed to quantitatively assess ORN activity:

In practice, researchers have assessed ORN activity by:

• Testing mitochondrial extracts from cells overexpressing ORN/REXO2 compared to controls

• Analyzing the degradation of specific substrates like pGpG in recombinant enzyme preparations

• Using substrate analogs such as pNP-TMP in structural and functional studies

Activity assays should include appropriate controls for substrate stability, enzyme specificity, and reaction conditions optimization (pH, temperature, divalent cations, salt concentration).

What genetic approaches are most effective for studying ORN function in bacterial systems?

Several genetic approaches have proven effective for studying ORN function:

• Conditional expression systems:

  • Anhydrotetracycline (Atc)-inducible promoter PLtetO-1 with Tet-repressor (TetR)

  • Lambda Red-assisted recombination for chromosomal integration

  • Verification via PCR of 5' (432 bp fragment) and 3' (446 bp fragment) integration sites

• Full deletion with plasmid complementation:

  • "Full deletion of orn in a clean genetic background"

  • "Controlled expression through tightly-regulated expression plasmids"

  • Allows titration of ORN levels to determine minimal requirements

• Double knockout studies:

  • Identifying synthetic lethal interactions (e.g., with RNase T or PNPase)

  • Reveals backup mechanisms and functional redundancy

• Site-directed mutagenesis:

  • Targeting catalytic residues to create activity-deficient variants

  • Separating different functions (e.g., pGpG degradation vs. general nanoRNA degradation)

• Reporter fusions:

  • Transcriptional/translational fusions to monitor expression regulation

  • Fluorescent protein tagging for localization studies

These approaches can be combined to provide comprehensive insights into ORN function, regulation, and interactions with other cellular components.

How can researchers isolate and analyze nanoRNAs to study ORN substrates?

• Specialized extraction protocols:

  • Modified RNA extraction methods with optimized for small RNA recovery

  • Size-exclusion filtration to enrich for very small RNA species

  • Avoid standard RNA precipitation methods that may lose small fragments

• Analytical techniques:

  • High-resolution polyacrylamide gel electrophoresis (PAGE) with appropriate markers

  • Liquid chromatography coupled with mass spectrometry (LC-MS)

  • Capillary electrophoresis for separation of oligoribonucleotides

• Enrichment strategies:

  • 3' adaptor ligation followed by reverse transcription

  • Size-selective precipitation methods

  • Ion-pair reversed-phase HPLC

• Comparative analysis:

  • Profile nanoRNAs in wild-type vs. ORN-deficient strains

  • Identify accumulating species in ORN mutants

  • Correlation with physiological changes (e.g., biofilm formation)

• Synthetic standards:

  • Use chemically synthesized oligoribonucleotides as standards

  • Include internal controls for extraction efficiency

  • Develop calibration curves for quantification

Research on P. aeruginosa demonstrated that Δorn cells possessed "highly elevated pGpG levels" , providing evidence of ORN's in vivo substrate. Similar approaches can be used to identify other physiologically relevant substrates in different bacterial species.

What experimental approaches reveal ORN's role in c-di-GMP signaling pathways?

Understanding ORN's role in c-di-GMP signaling requires multiple experimental approaches:

• Genetic manipulation:

  • Generation of Δorn mutants in model organisms like P. aeruginosa

  • Construction of conditional expression systems

  • Creation of catalytically inactive variants through site-directed mutagenesis

• Biochemical analysis:

  • In vitro assays measuring pGpG degradation by purified ORN

  • C-di-GMP degradation assays in cell lysates with/without ORN

  • Testing inhibition of EAL-dependent PDEs (PA2133, PvrR, RocR) by pGpG

• Metabolite quantification:

  • LC-MS measurement of intracellular c-di-GMP levels in wild-type vs. Δorn mutants

  • Quantification of pGpG accumulation in ORN-deficient cells

  • Time-course analysis of metabolite levels after stimulation

• Phenotypic characterization:

  • Biofilm formation assays (increased in Δorn mutants)

  • Extracellular polymer production assessment

  • Motility assays (swimming, swarming, twitching)

• Rescue experiments:

  • Addition of purified ORN to cell lysates to alleviate pGpG-dependent inhibition of PDEs

  • Complementation of Δorn mutants with plasmid-expressed ORN

  • Testing ORN orthologs from different species for functional conservation

These approaches collectively established that ORN "provides homeostatic control of intracellular pGpG under native physiological conditions and that this activity is fundamental to c-di-GMP signal transduction" .

How can recombinant ORN be utilized as a research tool in molecular biology?

Recombinant ORN offers several potential applications as a research tool:

• RNA sample preparation:

  • Removal of small RNA contaminants from RNA preparations

  • Cleanup of reaction products in RNA synthesis applications

  • Degradation of primers after reverse transcription

• Structural and functional RNA studies:

  • Investigation of nanoRNA roles in transcription priming

  • Studies on RNA degradation pathways

  • Analysis of small RNA regulatory functions

• c-di-GMP signaling research:

  • Modulation of pGpG levels in cell-free systems

  • Controlled degradation of signaling intermediates

  • Isolation of specific pathway components

• Analytical applications:

  • End-labeling of RNA by removing 3' terminal nucleotides

  • Generation of defined-length RNA fragments

  • Quality control tool for synthetic RNA

• Diagnostic potential:

  • Detection of specific short RNA species

  • Component in RNA amplification technologies

  • Possible applications in RNA-based biosensors

To implement these applications, researchers would need highly purified, characterized recombinant ORN with established activity parameters, stability profiles, and defined reaction conditions.

What insights has ORN research provided into bacterial RNA decay pathways?

Research on ORN has significantly advanced our understanding of bacterial RNA decay pathways:

• Final step in RNA degradation:

  • ORN performs the essential final step in RNA decay by converting oligoribonucleotides to monoribonucleotides

  • This function is critical for completing the RNA degradation process

• Redundancy and backup mechanisms:

  • Cellular viability depends on either RNase T or PNPase functioning in conjunction with ORN

  • This reveals redundancy in the final stages of RNA decay pathways

• Impact on small RNA metabolism:

  • Accumulation of nanoRNAs in ORN-deficient cells affects cellular processes

  • These small RNAs can prime transcription, potentially affecting global gene expression patterns

• Regulation of RNA processing:

  • ORN research revealed connections to RppH (RNA pyrophosphohydrolase) function

  • Studies showed that "absence of RppH does not affect the processing of tRNAs by RNase E, contrary to long-held beliefs"

• Evolutionary conservation:

  • High conservation of ORN across diverse bacterial species underscores the fundamental importance of complete RNA degradation

  • Presence of homologs in eukaryotes (REXO2) indicates ancient evolutionary origins of this function

These insights highlight the complexity and importance of RNA turnover in bacterial cells and the critical role that degradation of the smallest RNA fragments plays in cellular homeostasis.

What is the significance of ORN in biofilm formation and bacterial pathogenesis?

ORN plays a significant role in biofilm formation and potentially in bacterial pathogenesis through several mechanisms:

• Regulation of c-di-GMP signaling:

  • ORN degrades pGpG, preventing its accumulation and subsequent inhibition of EAL-dependent PDEs

  • This regulation helps maintain appropriate c-di-GMP levels

  • C-di-GMP is a master regulator of transitions between motile and biofilm lifestyles

• Impact on biofilm formation:

  • P. aeruginosa Δorn mutants exhibit high intracellular c-di-GMP levels

  • This causes "overexpression of extracellular polymers and overproduce biofilm"

  • Biofilms contribute to antibiotic resistance and persistent infections

• Potential virulence connections:

  • C-di-GMP regulates numerous virulence factors in pathogenic bacteria

  • ORN's influence on this signaling pathway could affect expression of virulence determinants

  • Biofilm formation itself is a significant virulence mechanism in many chronic infections

• Essential nature:

  • ORN is essential for viability in many bacteria

  • This essentiality makes it a potential target for antimicrobial development

  • Understanding ORN function could inform strategies to disrupt bacterial persistence

• RNA metabolism influence:

  • Changes in RNA turnover due to ORN dysfunction could affect expression patterns of virulence genes

  • Accumulation of nanoRNAs might alter stress responses important during infection

These connections highlight ORN as an important factor in bacterial adaptation and persistence in host environments, with potential implications for treating biofilm-associated infections.

What role does the human ORN homolog (REXO2) play in mitochondrial function?

The human ORN homolog, REXO2, plays several crucial roles in mitochondrial function:

• Dual localization and distribution:

  • Present in both cytosolic and mitochondrial fractions

  • Within mitochondria, localized to both the intermembrane space and matrix

  • Majority appears to be in the intermembrane space rather than matrix

• Oligoribonuclease activity:

  • Degrades small single-stranded RNA fragments in mitochondria

  • Mitochondrial extracts from cells overexpressing REXO2 show higher oligoRNase activity

  • Activity correlates with REXO2 distribution across submitochondrial fractions

• Impact on mitochondrial structure:

  • REXO2 depletion by RNA interference causes a striking morphological phenotype

  • Affected cells show "a disorganized network of punctate and granular mitochondria"

• Effects on mitochondrial nucleic acids and protein synthesis:

  • Lack of REXO2 causes "substantial decrease of mitochondrial nucleic acid content"

  • Results in "impaired de novo mitochondrial protein synthesis"

• Structural organization:

  • Forms a homotetrameric complex of approximately 90-100 kDa

  • Consistent with four mature monomers of 24.4 kDa each

  • Structure verified by Blue Native PAGE analysis

These findings constitute "the first in vivo evidence for an oligoribonuclease activity in human mitochondria" and highlight REXO2's importance in maintaining proper mitochondrial structure and function, with potential implications for understanding mitochondrial diseases.

How might ORN function be targeted for potential antimicrobial development?

ORN's essential nature and unique functions make it a potential target for antimicrobial development:

• Target rationale:

  • Essential for viability in many bacteria

  • Highly conserved across bacterial species

  • Performs a unique function with limited redundancy

• Potential targeting strategies:

  • Direct inhibition of enzymatic activity through small molecule inhibitors

  • Disruption of protein-protein interactions in multimeric complexes

  • Interference with substrate binding

  • Destabilization of protein structure

• Combination approaches:

  • Targeting both ORN and backup mechanisms (RNase T, PNPase)

  • Simultaneous inhibition of multiple RNA degradation pathways

  • Combining with biofilm-disrupting agents

• Selectivity considerations:

  • Structural and functional differences between bacterial ORN and human REXO2

  • Potential for selective inhibition that spares the human enzyme

  • Subcellular localization differences may allow targeting of bacterial cytoplasmic enzyme

• Biofilm-related applications:

  • Paradoxically, ORN enhancement might reduce biofilm formation

  • Preventing pGpG accumulation could disrupt established biofilms through c-di-GMP pathway modulation

• Development workflow:

  • High-throughput screening for inhibitors using purified recombinant ORN

  • Structural studies to guide rational drug design approaches

  • Cellular assays to confirm target engagement and antibacterial activity

  • Validation in infection models

Further structural and mechanistic studies of ORN will be essential to inform these potential therapeutic approaches and to identify specific vulnerabilities that could be exploited for antimicrobial development.