Recombinant Lactococcus lactis subsp. cremoris Ribonuclease 3 (rnc)

What is the functional role of Ribonuclease III in Lactococcus lactis subsp. cremoris?

Ribonuclease III (RNase III), encoded by the rnc gene in Lactococcus lactis, serves as a critical endoribonuclease in RNA metabolism. According to experimental evidence, L. lactis RNase III (Lac-RNase III) fulfills two primary functions: processing of ribosomal RNAs (rRNAs) and regulation of polynucleotide phosphorylase (PNPase) levels. Complementation assays have demonstrated that Lac-RNase III can substitute for Escherichia coli RNase III not only in rRNA processing but also in the processing of messenger RNAs (mRNAs) . This functional conservation suggests evolutionary importance and demonstrates the enzyme's versatility in RNA processing mechanisms across bacterial species.

What are the established phenotypic effects of rnc gene deletion in L. lactis strains?

Deletion of the rnc gene in L. lactis strains results in several observable phenotypic changes. Primary effects include altered ribosomal RNA processing patterns, which can be visualized through gel electrophoresis of total cellular RNA. Secondary effects include growth rate reduction, abnormal cell morphology, and altered stress responses. At the molecular level, rnc deletion leads to dysregulation of numerous genes due to improper mRNA processing, particularly those involved in RNA metabolism. In natural transformation systems, disruption of RNA processing pathways through rnc deletion can potentially impact competence gene expression, although this connection requires further investigation in L. lactis strains such as KW2 that exhibit natural transformability .

What are the optimal conditions for cloning and expressing recombinant L. lactis RNase III?

For successful cloning and expression of recombinant L. lactis RNase III, researchers should follow this optimized protocol:

• Cloning Strategy: The complete rnc gene should be amplified from L. lactis subsp. cremoris genomic DNA using high-fidelity polymerase with primers containing appropriate restriction sites for directional cloning.

• Expression Systems:

  • For heterologous expression: E. coli BL21(DE3) with pET expression vectors works effectively when the rnc gene is codon-optimized.

  • For homologous expression: The nisin-inducible expression system (NICE) in L. lactis is recommended with the following parameters:

    Parameter	Optimal Condition
    Growth temperature	30°C
    Induction timing	Mid-log phase (OD600 = 0.5-0.7)
    Nisin concentration	1-10 ng/ml
    Expression time	3-5 hours

• Purification Method: Histidine-tagged RNase III can be effectively purified using immobilized metal affinity chromatography (IMAC) followed by size-exclusion chromatography to ensure high purity and activity.

Research has shown that the enzyme remains stable and active when stored at -80°C in buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, and 50% glycerol .

How can researchers establish a reliable enzymatic assay for detecting L. lactis RNase III activity?

To establish a reliable enzymatic assay for L. lactis RNase III activity, researchers should implement the following methodology:

• Substrate Preparation:

  • Synthesize a specific double-stranded RNA substrate through in vitro transcription.

  • Research indicates that substrates used for B. subtilis RNase III are effectively cleaved by Lac-RNase III and can serve as reliable activity indicators .

• Reaction Conditions:

  Component	Optimal Concentration
  Buffer	20 mM Tris-HCl (pH 7.5)
  Salts	50 mM NaCl, 10 mM MgCl₂
  Reducing agent	1 mM DTT
  RNA substrate	0.1-1 μM
  Enzyme	10-100 nM
  Temperature	37°C
  Incubation time	15-30 minutes

• Activity Detection:

  • Analyze reaction products using denaturing polyacrylamide gel electrophoresis (PAGE).

  • Visualize using ethidium bromide staining or incorporate fluorescent or radioactive labels in the substrate.

  • Quantify cleavage products using densitometry or phosphorimaging.

• Controls:

  • Include a heat-inactivated enzyme control (95°C for 10 minutes).

  • Run a parallel reaction with commercially available E. coli RNase III as a positive control.

  • Include a reaction with known inhibitors like high EDTA concentrations (20 mM).

This standardized assay allows for specific detection of endonucleolytic activity of Lac-RNase III in both L. lactis and E. coli crude extracts .

What approaches can be used to study the in vivo targets of L. lactis RNase III?

To comprehensively identify and characterize in vivo targets of L. lactis RNase III, researchers should employ a multi-faceted approach:

• Comparative Transcriptomics:

  • Perform RNA-seq analysis comparing wild-type and rnc deletion mutants of L. lactis.

  • Focus on transcripts showing differential abundance or altered 5' and 3' termini.

  • Analyze data using specialized algorithms designed to detect processing events rather than just expression changes.

• CLIP-seq (Cross-linking Immunoprecipitation followed by Sequencing):

  • Express tagged versions of RNase III (e.g., FLAG-tag or His-tag).

  • Cross-link protein-RNA complexes in vivo using UV irradiation.

  • Immunoprecipitate the enzyme-RNA complexes and sequence the bound RNA fragments.

  • Map sequences to the genome to identify binding sites.

• Structure Probing of Candidate Targets:

  • Perform in vitro structure analysis of potential target RNAs using techniques like SHAPE (Selective 2'-hydroxyl acylation analyzed by primer extension).

  • Compare cleavage patterns of wild-type and mutated versions of target RNAs.

• Validation Experiments:

  • Confirm direct cleavage by incubating purified RNase III with in vitro transcribed candidate RNA targets.

  • Perform site-directed mutagenesis of predicted cleavage sites in target RNAs.

  • Analyze the effects of these mutations on RNA processing and stability in vivo.

Implementation of these complementary approaches provides a comprehensive map of RNase III targets and their regulatory implications in L. lactis.

How should researchers interpret variations in RNase III activity between different L. lactis strains?

When interpreting variations in RNase III activity between different L. lactis strains, researchers should consider multiple factors that influence enzyme function:

• Genetic Sequence Variation Analysis:

  • Perform comparative genomics focusing on the rnc gene and its regulatory regions across strains.

  • Identify single nucleotide polymorphisms (SNPs) and correlate them with observed activity differences.

  • Pay particular attention to mutations affecting the catalytic domain or RNA-binding regions.

• Expression Level Assessment:

  • Quantify rnc transcript levels using RT-qPCR across different strains under identical growth conditions.

  • Measure protein levels using Western blotting with specific antibodies or tagged versions of the enzyme.

  • Create a correlation matrix between expression levels and measured enzymatic activity.

• Contextual Factors Evaluation:

  • Determine whether differences in cellular environments (pH, ion concentrations) contribute to activity variations.

  • Assess whether alternative RNA processing pathways may compensate for RNase III variations in certain strains.

  • Consider strain-specific co-factors that might enhance or inhibit RNase III activity.

• Functional Impact Assessment:

  • Compare rRNA and mRNA processing patterns across strains using Northern blotting or high-resolution RNA-seq.

  • Examine whether variations in RNase III activity correlate with strain-specific phenotypes such as growth rates, stress responses, or competence development .

This systematic approach allows researchers to distinguish between variations due to intrinsic enzyme differences versus contextual factors, providing insight into strain-specific RNA metabolism strategies.

How can researchers differentiate between direct and indirect effects when analyzing phenotypes of rnc deletion mutants?

Differentiating between direct and indirect effects in rnc deletion mutants requires a systematic approach:

• Temporal Analysis of Molecular Changes:

  • Implement time-course experiments after conditional depletion of RNase III.

  • Early changes (minutes to hours) likely represent direct effects on RNA processing.

  • Later changes (hours to days) typically reflect downstream consequences and adaptive responses.

  • Plot the temporal relationship between primary molecular events and subsequent phenotypic changes.

• Complementation Studies with Targeted Approaches:

  • Reintroduce wild-type rnc under inducible control to identify reversible phenotypes.

  • Introduce catalytically inactive RNase III mutants to distinguish between enzymatic and structural roles.

  • Use the following classification framework:

    Phenotype Response	Interpretation
    Immediate reversal with wild-type rnc	Likely direct effect
    Delayed reversal with wild-type rnc	Possible indirect effect
    Partial reversal with catalytic mutant	Potential scaffold function
    No reversal with any construct	Complex/multifactorial effect

• Target-Specific Rescue Experiments:

  • Identify putative direct RNase III targets via high-throughput methods.

  • Express these targets from alternative, RNase III-independent constructs.

  • Assess whether phenotypic effects can be rescued by manipulating individual targets.

• Network Analysis Integration:

  • Map observed changes onto known regulatory networks.

  • Identify network nodes that connect direct RNase III targets to observed phenotypes.

  • Calculate the minimum path length between direct targets and phenotypic effects.

This methodical approach allows researchers to construct a hierarchical model of RNase III-dependent effects, distinguishing proximal molecular events from distal phenotypic consequences.

How can L. lactis RNase III be utilized as a tool for genome editing in lactic acid bacteria?

L. lactis RNase III can be repurposed as a sophisticated genome editing tool for lactic acid bacteria through the following approaches:

• RNA-guided Genome Editing Systems:

  • Engineer chimeric RNA guides containing RNase III recognition motifs flanking target sequences.

  • Fuse RNase III to catalytically dead Cas9 (dCas9) to create a programmable RNA cleavage system.

  • The system can be designed to target specific mRNAs for degradation or to process precursor RNAs into functional forms.

• Recombination Enhancement:

  • Exploit RNase III's ability to process structured RNAs to enhance natural transformation efficiency in competent L. lactis strains like KW2 .

  • Design recombination templates with strategic secondary structures that, when processed by RNase III, expose homology regions more efficiently.

  • Implementation parameters:

    Parameter	Optimized Setting
    Template structure	Hairpin at termini
    RNase III level	2-3× normal expression
    Competence induction	Synchronized with RNase III expression
    Recombination efficiency	3-5× improvement over standard methods

• Conditional Gene Expression Control:

  • Develop synthetic RNA switches containing RNase III recognition sites.

  • In the presence of specific triggers (temperature, pH, metabolites), these RNA structures change conformation, becoming either more or less susceptible to RNase III cleavage.

  • This allows post-transcriptional regulation of gene expression without modifying transcriptional control elements.

• Multi-gene Operon Processing:

  • Design artificial operons with RNase III processing sites between genes.

  • Enable differential stability or translation efficiency of genes within a single transcription unit.

  • Particularly valuable for metabolic engineering applications requiring precise stoichiometric expression of pathway components.

These applications leverage the natural RNA processing capabilities of RNase III while expanding its utility for precise genetic manipulation of lactic acid bacteria.

What role might L. lactis RNase III play in modulating the host immune system when using L. lactis as a vaccine delivery platform?

L. lactis RNase III may play a significant role in modulating host immune responses when L. lactis is used as a vaccine delivery platform:

This multifaceted role makes RNase III activity an important consideration when engineering L. lactis as a vaccine delivery platform.

How can researchers exploit the substrate specificity of L. lactis RNase III for selective RNA targeting applications?

Researchers can leverage the unique substrate specificity of L. lactis RNase III for selective RNA targeting through several sophisticated approaches:

• Engineered RNA Decoys with Strain-Specific Recognition:

  • Design synthetic RNA molecules containing L. lactis RNase III recognition motifs.

  • These decoys can competitively inhibit the enzyme's activity on natural substrates.

  • Create libraries of decoy variants with systematic modifications to determine sequence and structural features that enhance specificity.

  • Validation assays should measure:

    • Binding affinity (Kd values) using surface plasmon resonance

    • Competitive inhibition constants (Ki) in enzymatic assays

    • In vivo efficacy using reporter systems

• Chimeric Ribozyme-RNase III Substrate Constructs:

  • Develop RNA molecules containing both RNase III recognition structures and ribozyme domains.

  • RNase III cleavage triggers conformational changes that activate the ribozyme.

  • This creates a two-step RNA processing system with enhanced specificity.

  • Applications include:

    • Conditional gene silencing systems

    • RNA circuitry with Boolean logic capabilities

    • Sensor-actuator systems responsive to cellular conditions

• Substrate Preference Profiling and Engineering:

  • Perform systematic analysis of natural substrates using the following workflow:

    Analysis Phase	Methodology	Output
    Discovery	CLIP-seq or RNA-seq	Comprehensive substrate inventory
    Feature extraction	Structural mapping	Common motifs and structures
    Preference testing	In vitro cleavage assays	Kinetic parameters
    Specificity engineering	Directed evolution	Enhanced targeting variants

  • Use this data to design RNA structures with tailored sensitivity to L. lactis RNase III.

• Cross-Species RNA Targeting:

  • Exploit the finding that Lac-RNase III can process substrates from other bacterial species like B. subtilis .

  • Design chimeric RNA structures combining recognition elements from multiple species.

  • Create selectivity profiles that distinguish between closely related bacterial RNase III enzymes.

  • Applications include:

    • Species-specific RNA therapeutics

    • Diagnostic tools for bacterial identification

    • Selective targeting within mixed microbial communities

This strategic exploitation of substrate specificity opens new avenues for both fundamental research and biotechnological applications involving targeted RNA processing.

What strategies can overcome low expression yields of recombinant L. lactis RNase III?

When facing low expression yields of recombinant L. lactis RNase III, researchers should implement the following troubleshooting strategies:

• Optimization of Expression Parameters:

  • Systematically vary induction conditions using this experimental matrix:

    Parameter	Test Range	Optimal Range for L. lactis RNase III
    Temperature	16-37°C	25-30°C (reduced proteolysis)
    Inducer concentration	0.1-2 mM IPTG (E. coli) or 0.1-50 ng/ml nisin (L. lactis)	0.5 mM IPTG or 10 ng/ml nisin
    OD600 at induction	0.3-1.2	0.6-0.8 (mid-log phase)
    Post-induction time	2-24 hours	4-6 hours (balance between yield and activity)

• Codon Optimization and Expression Construct Redesign:

  • Analyze the rnc coding sequence for rare codons using strain-specific codon usage tables.

  • Optimize the sequence while preserving any critical regulatory secondary structures.

  • Adjust the ribosome binding site strength using predictive algorithms.

  • Include solubility-enhancing fusion partners (e.g., MBP, SUMO) that can be later removed by specific proteases.

• Host Strain Engineering:

  • For E. coli expression: Use specialized strains like Rosetta (supplies rare tRNAs) or Arctic Express (enhanced folding at low temperatures).

  • For L. lactis expression: Consider protease-deficient strains or co-expression of chaperones.

  • Implement transient inhibition of host RNases during induction to prevent degradation of overexpressed rnc mRNA, as research shows E. coli RNase III can trigger degradation of heterologous rnc mRNA .

• Process Optimization:

  • Implement fed-batch cultivation to reduce acetate formation and extend productive growth phase.

  • Add stabilizing agents like glycerol (5-10%) or specific metal ions (Mg2+, Mn2+) to the culture medium.

  • For difficult cases, consider cell-free protein synthesis systems with optimized redox conditions.

These strategies address issues at different levels of the expression process, from transcription to protein folding and stability, enabling systematic troubleshooting of yield problems.

How can researchers address challenges in purifying active L. lactis RNase III without contaminating nucleases?

Purifying active L. lactis RNase III without nuclease contamination requires a strategic approach addressing multiple purification challenges:

• Optimized Expression Systems and Initial Processing:

  • Express the protein with an N-terminal His-tag to avoid interference with the C-terminal catalytic domain.

  • Include a TEV protease cleavage site between the tag and the protein for tag removal after purification.

  • Process harvested cells using this protocol to minimize nuclease activation:

    • Resuspend in buffer containing 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5% glycerol, 1 mM DTT, and 5 mM EDTA

    • Add nuclease inhibitors: 1 mM PMSF, 0.5 μg/ml leupeptin, and 2 U/ml DNase-free RNasin

    • Lyse cells by sonication (5 cycles of 30 seconds on/30 seconds off) at 4°C

    • Clarify by centrifugation at 20,000 × g for 30 minutes at 4°C

• Multi-stage Chromatography Strategy:

  • Implement a three-stage purification scheme:

    Purification Stage	Method	Purpose	Critical Parameters
    Capture	IMAC (Ni-NTA)	Initial purification	20 mM imidazole in wash buffer to reduce non-specific binding
    Intermediate	Heparin affinity	Remove contaminating nucleases	0.5-1.0 M NaCl gradient elution
    Polishing	Size exclusion	Separate aggregates and final contaminants	Pre-equilibrated with RNase-free buffer

• Contamination Testing and Resolution:

  • Implement sensitive nuclease contamination assays:

    • Incubate purified protein with specific RNA and DNA substrates

    • Analyze degradation patterns using denaturing PAGE

    • Distinguish RNase III-specific cleavage (producing characteristic fragments) from non-specific degradation

  • If contamination persists:

    • Implement additional purification steps like ion exchange chromatography

    • Consider activity-based separation using substrate affinity columns

    • Explore refolding from inclusion bodies as an alternative purification strategy

• Stabilization of Purified Enzyme:

  • Store the final preparation in buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, and 50% glycerol

  • Add RNase inhibitor (1 U/μl) to inhibit any residual contaminating RNases

  • Aliquot and flash-freeze in liquid nitrogen

  • Store at -80°C for maximum stability

This comprehensive approach addresses the specific challenges of purifying active RNase III while minimizing contamination with other nucleases that would interfere with subsequent applications.

What controls should be included when studying the effects of L. lactis RNase III on immune modulation?

When investigating the effects of L. lactis RNase III on immune modulation, researchers should implement a comprehensive set of controls to distinguish direct enzyme effects from other bacterial factors:

• Genetic Controls:

  • Wild-type L. lactis strain (baseline immune response)

  • RNase III deletion mutant (Δrnc) (absence of enzymatic activity)

  • RNase III complemented strain (restoration of phenotype)

  • Catalytically inactive RNase III mutant (distinguishes between structural and enzymatic effects)

  • Control for secretion system effects (if using secretion-based delivery)

• Bacterial Component Controls:

  • Purified recombinant L. lactis RNase III (direct enzyme effects)

  • Heat-inactivated L. lactis cells (distinguishes between active and passive mechanisms)

  • Cell wall fraction (controls for TLR2-mediated effects)

  • Cytoplasmic fraction (controls for internal bacterial components)

  • RNA/DNA fractions (controls for nucleic acid-mediated signaling)

• Immune Cell Validation Series:

  • Implement this testing matrix across different immune cell types:

    Cell Type	Readouts	Purpose	Relevant Controls
    Dendritic cells	CD11c+, ALDH+, gene expression (Aldh1a2, Itgav, Itgb8, Il10)	Assess antigen presentation modulation	TLR-deficient DCs (e.g., TLR2-/- MLN DCs)
    T cells	Foxp3+, IL-10+ subpopulations	Measure T cell differentiation	OVA-specific transgenic T cells with OVA peptide
    Macrophages	M1/M2 polarization markers	Assess macrophage polarization	LPS-stimulated cells
    Epithelial cells	Barrier integrity, cytokine production	Measure effects on mucosal barrier	Transwell cultures with TEER measurement

• Systems-level Controls:

  • In vitro co-culture systems with varying components:

    • DCs alone with L. lactis variants

    • DC-T cell co-cultures with antigen and L. lactis variants

    • Triple cultures including epithelial cells

  • In vivo models with appropriate controls:

    • Germ-free versus conventional mice

    • Adoptive transfer of specific cell populations

    • Receptor knockout models (e.g., TLR-deficient mice)

Research has demonstrated that L. lactis strains can modulate dendritic cell function, with effects on regulatory T cell induction . These comprehensive controls will help distinguish whether RNase III directly contributes to these immunomodulatory properties through RNA processing or indirectly through effects on bacterial fitness and gene expression.

How might L. lactis RNase III be engineered for enhanced specificity in RNA targeting applications?

Engineering L. lactis RNase III for enhanced specificity in RNA targeting applications presents several promising avenues for research:

• Structure-guided Mutagenesis:

  • Target the RNA-binding domain using a systematic approach:

    • Identify conserved residues through multiple sequence alignment across bacterial RNase III enzymes

    • Perform alanine scanning mutagenesis of the RNA-binding interface

    • Introduce amino acid substitutions that alter charge distribution or hydrophobicity

  • Expected outcomes include variants with:

    • Altered substrate length requirements

    • Modified sequence preferences at cleavage sites

    • Differential activity on structured versus unstructured RNAs

• Domain Swapping and Chimeric Enzymes:

  • Create chimeric RNase III enzymes by combining domains from different bacterial species:

    • Replace the substrate recognition domain while maintaining the catalytic domain

    • Engineer the linker regions to optimize spatial orientation between domains

  • Potential domain combinations include:

    Donor Species	Domain	Expected Benefit
    E. coli	dsRNA binding domain	Enhanced binding to canonical substrates
    B. subtilis	N-terminal domain	Altered substrate specificity profile
    Streptococcus	Catalytic domain	Modified cleavage pattern
    Lactococcus	Full enzyme	Baseline for comparison

• Fusion Protein Approaches:

  • Develop fusion proteins combining RNase III with heterologous RNA-binding domains:

    • PUF domains (programmable RNA binding with 8-nt specificity)

    • CRISPR-derived dCas9 or Cas13 (RNA-guided targeting)

    • MS2 coat protein (recognition of specific RNA stem-loops)

  • Design considerations include:

    • Flexible linker optimization for spatial freedom

    • Inducible dimerization domains for activity control

    • Split enzyme approaches for binary logic operations

• Directed Evolution Strategies:

  • Implement directed evolution using:

    • Error-prone PCR to generate variant libraries

    • Selection systems based on RNA cleavage-dependent gene expression

    • Deep sequencing to identify beneficial mutations

  • Activity screening platforms:

    • Two-hybrid systems linking RNA cleavage to reporter expression

    • In vitro compartmentalization for high-throughput activity screening

    • Cell survival linked to specific RNA targeting

These engineering approaches could yield L. lactis RNase III variants with enhanced specificity for applications in synthetic biology, gene therapy, and precision biotechnology. The resulting enzymes may enable selective targeting of structural RNA motifs associated with specific genes or pathogens.

What potential synergies exist between L. lactis RNase III and CRISPR-Cas systems for genome editing applications?

Exploring the synergies between L. lactis RNase III and CRISPR-Cas systems reveals several promising avenues for enhanced genome editing applications:

• RNA Processing Enhancement of CRISPR Efficiency:

  • RNase III plays a critical role in processing CRISPR array transcripts in many bacteria.

  • Optimizing RNase III activity could enhance guide RNA maturation in engineered CRISPR systems.

  • Research potential effects on efficiency:

    RNase III Modification	Potential Impact on CRISPR System	Mechanism
    Overexpression	Increased guide RNA processing rate	Enhanced maturation of crRNA
    Specificity engineering	Improved precision of guide RNA generation	Reduced off-target effects
    Temporal control	Synchronized guide RNA availability	Better editing kinetics
    Co-localization with Cas proteins	Streamlined processing-to-editing pipeline	Improved molecular assembly

• Dual Nuclease Systems for Complex Editing:

  • Combine DNA-targeting Cas9 with RNA-targeting RNase III for simultaneous genomic and transcriptomic editing.

  • Applications include:

    • Concurrent gene knockout and mRNA regulation

    • Combined DNA repair template delivery with transient mRNA knockdown

    • Creation of genetic/epigenetic feedback loops

• Natural Transformation Enhancement:

  • Exploit the functional natural transformation system in L. lactis subsp. cremoris KW2 .

  • Modulate RNase III activity to enhance the competence state by:

    • Processing competence gene mRNAs to optimize expression

    • Regulating stability of transforming DNA after uptake

    • Modifying cell envelope components through downstream gene regulation

  • Combine with CRISPR selection systems for markerless editing

• RNA-guided RNase III Targeting:

  • Create chimeric systems combining:

    • CRISPR RNA-binding capabilities (from Cas13 or dCas9-RNA tethering)

    • RNase III catalytic activity

  • This fusion approach offers:

    • Programmable RNA targeting beyond natural RNase III substrates

    • Potential for multiplexed RNA processing

    • Reduced off-target effects compared to Cas13

    • Options for regulating gene expression through selective mRNA processing rather than degradation

The combination of these two systems—CRISPR-Cas and RNase III—offers complementary capabilities that could address current limitations in genome editing efficiency, specificity, and delivery, particularly in lactic acid bacteria where established genetic tools are more limited than in model organisms.

How could high-throughput methodologies be applied to comprehensively map the RNase III degradome in L. lactis?

Mapping the complete RNase III degradome in L. lactis requires integration of cutting-edge high-throughput methodologies:

• Global RNA End Mapping Technologies:

  • Implement RNA-seq variants designed to capture specific RNA termini:

    • SPARE (Sequencing Polyadenylated RNA 5' Ends): Captures 5' ends generated by RNase cleavage

    • PARE (Parallel Analysis of RNA Ends): Identifies uncapped 5' ends resulting from endonucleolytic cleavage

    • 3'-RACE-seq: Maps 3' termini genome-wide

  • Analytical workflow:

    • Compare wild-type and Δrnc strains under identical conditions

    • Identify ends present in wild-type but absent in mutant

    • Quantify cleavage efficiency at each site

    • Map positions to genomic features (UTRs, coding sequences, non-coding RNAs)

• CLIP-seq and Variants for Direct Binding Site Identification:

  • Apply CLIP-seq (Cross-linking and Immunoprecipitation followed by sequencing) using:

    • FLAG- or His-tagged RNase III expressed at near-native levels

    • UV crosslinking to capture direct RNA-protein interactions

    • RNase footprinting to identify protected regions

  • Enhanced variants:

    • iCLIP (individual nucleotide resolution)

    • PAR-CLIP (photoactivatable ribonucleoside-enhanced)

    • CLASH (crosslinking, ligation, and sequencing of hybrids) to identify RNA-RNA interactions at cleavage sites

• Parallel Substrate Validation Platform:

  • Design a massively parallel reporter system:

    • Synthesize library of thousands of potential RNase III substrates with systematic variations

    • Incorporate into a reporter system where cleavage modulates expression of fluorescent protein

    • Analyze through flow cytometry and next-generation sequencing

  • Expected output:

    Analysis Type	Methodology	Information Gained
    Sequence motifs	k-mer enrichment analysis	Primary sequence preferences
    Structural features	RNA folding algorithms	Secondary structure requirements
    Kinetic parameters	Time course analysis	Processing efficiency metrics
    Context effects	Positional analysis	Impact of surrounding sequences

• Integrative Multi-omics Approach:

  • Combine multiple data types:

    • RNase III-CLIP data (direct binding)

    • RNA-seq (abundance changes)

    • RNA end mapping (cleavage sites)

    • Ribosome profiling (translation effects)

  • Integration strategy:

    • Develop computational pipeline correlating all features

    • Apply machine learning to identify complex patterns

    • Generate predictive models for novel substrate identification

    • Create accessible database of the L. lactis RNase III degradome

This comprehensive approach would generate an unprecedented view of RNase III function in L. lactis, revealing not only direct targets but also processing kinetics, regulatory networks, and evolutionary conservation patterns of this essential ribonuclease.