Recombinant Oryza sativa subsp. japonica tRNA-dihydrouridine (47) synthase [NAD (P) (+)]-like (Os04g0117600, LOC_Os04g02730), partial

What is the function of tRNA-dihydrouridine synthase in Oryza sativa and how does it compare to other organisms?

tRNA-dihydrouridine synthase (DUS) in Oryza sativa catalyzes the synthesis of dihydrouridine, a modified base found in the D-loop of most tRNAs . This enzyme belongs to the C3H family of proteins and contains a CCCH-type zinc finger domain along with a tRNA-dihydrouridine synthase domain .

Unlike thermophilic bacteria such as Thermus thermophilus which has only one DUS gene encoding an enzyme that catalyzes just two D modifications (D20 and D20a) , eukaryotes like O. sativa possess multiple DUS enzymes. This suggests a higher complexity of tRNA modification in rice compared to extremophiles.

The rice DUS enzyme specifically belongs to the DUS3 subfamily, as indicated by database annotations . Comparative studies have shown that human DUS2 exclusively modifies U20 across diverse tRNA substrates , and similar site-specific activity is likely for rice DUS proteins, though with potentially different target positions.

What genomic and structural features characterize the Os04g0117600/LOC_Os04g02730 gene and protein?

The LOC_Os04g02730 gene encodes a 697 amino acid protein with a molecular weight of approximately 77.5 kDa and an isoelectric point of 7.78 . The protein contains several key domains:

• A zinc finger CCCH-type domain (amino acids 98-129)

• An aldolase-type TIM barrel (amino acids 340-583)

• A tRNA-dihydrouridine synthase domain (amino acids 347-581)

• A conserved tRNA-dihydrouridine synthase site (amino acids 428-446)

The gene has multiple transcript variants, with at least three isoforms identified (X1, X2, and X3) . Its promoter is active in various tissues including leaves, embryos, roots, panicles, and ovaries, indicating widespread expression throughout the plant .

The protein features NAD(P)+ binding capability through a flavin adenine dinucleotide binding domain , which is essential for its enzymatic function in catalyzing the reduction of uridine to dihydrouridine.

What expression patterns does Os04g0117600/LOC_Os04g02730 exhibit across rice tissues and developmental stages?

Based on transcriptomic data, LOC_Os04g02730 shows preferential expression in green tissues. RT-qPCR analysis of the PDD gene (a homologous tRNA-modifying enzyme in rice) revealed highest expression in:

• Leaves at the seedling stage

• Leaves and sheaths at the tillering stage

• Much lower expression in roots, stems, and panicles

GUS reporter gene studies driven by the promoter of this gene showed activity in:

• Leaves

• Embryos of seeds

• Roots

• Panicles

• Ovaries

Expression data from UniGene indicates that the transcript has been detected in multiple tissues including callus, flower, leaf, panicle, root, stem, and vegetative meristem , suggesting a ubiquitous but tissue-variable expression pattern.

How are tRNA genes distributed in the Oryza sativa genome, and what implications does this have for dihydrouridine synthase function?

The O. sativa genome contains approximately 750 tRNA genes distributed unevenly across different chromosomes . These encode 52 different isoacceptors, with tRNA Ser having the highest abundance of isoacceptors (5 different types: GCT, TGA, AGA, CGA, and GGA) .

Key characteristics of rice tRNA distribution:

• tRNA genes range from 66 to 91 nucleotides in length

• Chromosome 8 contains the smallest known tRNA in eukaryotes (tRNA Ser with only 66 nucleotides)

• Multiple novel/pseudo tRNA genes have been identified

• Family-specific conserved consensus sequences are present

Approximately 4.53% (34 out of 750) of rice tRNAs contain introns, with tRNA Met having the highest number of intron-containing genes (15 out of 60, or 25%) . The presence of these diverse tRNA structures suggests that rice DUS enzymes must recognize and process a variety of substrates, potentially requiring sophisticated recognition mechanisms beyond simple sequence motifs.

What methodologies can be employed to analyze substrate specificity of rice tRNA-dihydrouridine synthase?

Several complementary approaches can be utilized to determine substrate specificity of rice DUS:

Mechanism-based crosslinking:
This approach, successfully used with human DUS2, involves creating 5-bromouridine (5-BrUrd)-modified oligonucleotide probes that crosslink with the enzyme during the catalytic process . For rice DUS:

• Design 5-BrUrd-modified tRNA probes based on known rice tRNA sequences

• Express and purify recombinant rice DUS protein

• Incubate the enzyme with the probes under various conditions

• Analyze crosslinking efficiency using gel electrophoresis and mass spectrometry

In vitro dihydrouridylation assays:

• Express and purify recombinant rice DUS without bound tRNA

• Synthesize or isolate various tRNA substrates from rice

• Incubate enzyme with tRNA under physiologically relevant conditions

• Analyze modification using liquid chromatography-coupled tandem quadrupole mass spectrometry (LC-MS/MS)

Comparative studies with mutated tRNAs:
Based on research with human DUS2, which identified a minimal GU motif within the tRNA tertiary fold required for directing activity , researchers could:

• Create a series of mutated rice tRNAs with alterations in potential recognition motifs

• Perform in vitro dihydrouridylation assays on these variants

• Determine the minimal sequence requirements for substrate recognition

What techniques are most effective for expressing and purifying active recombinant rice tRNA-dihydrouridine synthase?

Based on successful purification of DUS enzymes from various organisms, the following protocol is recommended:

Expression system selection:

• E. coli BL21(DE3) with pET-based vectors for basic characterization

• Yeast expression systems (S. cerevisiae) for more complex eukaryotic post-translational modifications, as used successfully for TYW proteins

• Insect cell expression for higher eukaryotic protein folding capabilities

Purification strategy:

• Use an N-terminal or C-terminal hexahistidine tag for initial IMAC (immobilized metal affinity chromatography) purification

• Follow with size exclusion chromatography to obtain monomeric protein

• Verify flavin cofactor incorporation through spectroscopic analysis (absorbance at 450 nm)

Activity preservation:

• Add 10-15% glycerol to storage buffers to prevent protein aggregation

• Include reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to maintain cysteine residues

• Store at -80°C in small aliquots to avoid freeze-thaw cycles

Activity assessment:

• Verify enzyme functionality using in vitro assays with model tRNA substrates

• Monitor cofactor binding by fluorescence spectroscopy

• Assess thermal stability through thermal shift assays or circular dichroism

How can mass spectrometry be optimized for detection and quantification of dihydrouridine modifications in rice tRNAs?

Mass spectrometry has been successfully used to detect dihydrouridine modifications in tRNAs. For rice-specific applications:

Sample preparation:

• Extract total RNA from rice tissues using TRIzol or similar methods

• Enrich for tRNAs using size exclusion or affinity chromatography

• Digest tRNAs with specific nucleases (RNase T1 for G-specific cleavage)

• Dephosphorylate RNA fragments if necessary

LC-MS/MS analysis parameters:

• Use porous graphitic carbon or C18 reverse-phase chromatography for nucleoside separation

• Employ multiple reaction monitoring (MRM) for targeted detection of dihydrouridine

• Monitor the molecular transitions specific to dihydrouridine (m/z 247→115)

Quantification approach:

• Use stable isotope-labeled internal standards for absolute quantification

• Apply ion-pairing agents (e.g., triethylamine) to improve separation

• Implement parallel reaction monitoring for improved selectivity

Data analysis strategy:
Analyze data using the "ribonucleome analysis" approach as described for yeast DUS enzymes :

• Compare mass of anticodon-containing fragments from wild-type and mutant samples

• Look for mass shifts corresponding to the absence of dihydrouridine

• Quantify modification stoichiometry by comparing modified and unmodified forms

What are the known sequence and structural determinants for selective dihydrouridylation by dihydrouridine synthases?

Research on human and yeast DUS enzymes has revealed several key determinants for site-specific dihydrouridylation that may apply to rice DUS:

Sequence determinants:

• A minimal GU motif within the tRNA tertiary fold is critical for hDUS2 activity targeting U20

• Yeast DUS enzymes show non-overlapping substrate sites in the D-loop:

  • Dus1p modifies U16/17

  • Dus2p modifies U20

  • Dus4p modifies U20a/20b

Structural requirements:

• The tertiary structure of tRNA is essential for proper substrate recognition

• Bacterial DUS-tRNA interactions show that residues U16 and U20, located on opposite sides of the D loop, are selectively recognized through reorientation of tRNA binding

• Studies on bacterial P. aeruginosa DusA reveal specific structural features involved in substrate binding

Recognition mechanisms:
Research suggests that DUS enzymes recognize their substrates through:

• Specific interactions with the tRNA tertiary fold

• Recognition of nucleotides adjacent to the target uridine

• Protein-induced conformational changes in tRNA structure

Understanding these determinants in rice DUS would require:

• Structural biology approaches (X-ray crystallography, cryo-EM)

• Mutagenesis studies of both enzyme and substrate

• Computational modeling of enzyme-substrate interactions

What are the physiological impacts of dihydrouridine modification in rice tRNAs and how can they be experimentally assessed?

Dihydrouridine modifications impact tRNA structure and function in several ways that can be experimentally assessed:

Physiological impacts:

• Stabilization of tRNA structure, particularly in psychrophiles

• Enhanced flexibility of tRNA, especially in the D-loop region

• Influence on translation efficiency and accuracy

• Potential role in stress response mechanisms

Experimental assessment approaches:

• Gene disruption studies:

  • Generate CRISPR/Cas9 knockout or knockdown rice lines lacking functional DUS genes

  • Assess phenotypic changes under normal and stress conditions

  • Analyze changes in protein synthesis rates using metabolic labeling

• Ribosome profiling:

  • Compare translation efficiency in wild-type vs. DUS-deficient rice

  • Identify specific mRNAs affected by altered tRNA modification

• Structural analysis:

  • Employ chemical probing to assess tRNA structural differences

  • Use nuclear magnetic resonance (NMR) to analyze tRNA conformational dynamics

• Physiological stress experiments:

  • Subject wild-type and DUS-deficient rice to various stresses (temperature, salinity, drought)

  • Quantify changes in dihydrouridine levels under different conditions

  • Correlate modification levels with physiological responses

• Chloroplast protein synthesis assessment:
  Based on studies showing that PDD (a tRNA-modifying enzyme) affects chloroplast function :

  • Analyze chloroplast protein levels using proteomics

  • Monitor photosynthetic efficiency in DUS-deficient plants

  • Assess ribosome biogenesis in chloroplasts

What computational approaches can predict potential substrate tRNAs and modification sites for rice dihydrouridine synthase?

Several computational approaches can be employed to predict tRNA substrates and modification sites:

Sequence-based prediction methods:

• Position-specific scoring matrices (PSSMs) based on known modification sites

• Machine learning algorithms trained on experimentally verified dihydrouridylation sites

• Conservation analysis across species to identify likely substrate recognition patterns

Structure-based prediction approaches:

• Homology modeling of rice DUS based on available crystal structures

• Molecular docking of tRNA structures with the modeled enzyme

• Molecular dynamics simulations to analyze enzyme-substrate interactions

Integration with experimental data:
For rice DUS, researchers could:

• Start with known modification sites from related species (yeast, human)

• Apply conservation analysis to rice tRNA genes

• Use machine learning to predict likely modification sites

• Validate predictions with targeted experiments

Prediction pipeline example:

• Extract all rice tRNA sequences from genomic data

• Predict their secondary and tertiary structures

• Identify potential U20 positions (based on human DUS2 specificity)

• Analyze flanking sequences for GU motifs identified in human studies

• Calculate accessibility scores for target uridines

• Rank tRNAs by likelihood of being substrates

How does temperature affect the dihydrouridylation activity of rice tRNA-dihydrouridine synthase compared to enzymes from other species?

Temperature significantly impacts dihydrouridine synthase activity across species, with important implications for rice research:

Comparative temperature effects:

• Thermophilic bacteria like T. thermophilus show reduced dihydrouridine content in tRNAs compared to mesophiles

• The thermophilic T. thermophilus Dus successfully modifies tRNA^Phe transcript at 60°C, demonstrating thermostability

• In T. thermophilus, other tRNA modifications are required for D formation at high temperatures

Experimental approach for rice DUS temperature dependency:

• Express and purify recombinant rice DUS enzyme

• Perform in vitro dihydrouridylation assays at temperature ranges (10-50°C)

• Determine thermal stability using circular dichroism or differential scanning fluorimetry

• Compare thermal melting point (T₍m₎) of rice DUS to enzymes from thermophiles and psychrophiles

A thermal stability study of P. aeruginosa DusA showed a T₍m₎ value of 46.2°C and chemical denaturation midpoint (C₍m₎) of 2.7 M for urea . Similar studies on rice DUS would provide valuable comparative data.

Predictive analysis:
Based on ecological adaptation, rice DUS enzymes would likely show optimal activity between 25-35°C, corresponding to typical rice growing conditions. Higher temperatures may lead to protein destabilization and reduced activity, while lower temperatures might decrease catalytic efficiency.

What are the current challenges and solutions in studying the relationship between dihydrouridine modification and rice stress responses?

Studying dihydrouridine modifications in relation to rice stress response presents several challenges and potential solutions:

Challenges:

• Low abundance of modified nucleosides:
  Dihydrouridine represents a small fraction of total RNA, making detection difficult.

• Tissue-specific and developmental regulation:
  Modification levels may vary across tissues and growth stages, requiring detailed temporal and spatial analysis.

• Multiple DUS enzymes with overlapping functions:
  Rice likely has several DUS enzymes with potentially redundant activities.

• Environmental variability:
  Field conditions introduce numerous variables affecting tRNA modification.

• Distinguishing cause from effect:
  Determining whether changes in modification levels cause stress responses or result from them.

Methodological solutions:

• Sensitive detection methods:

  • Develop targeted LC-MS/MS methods with improved sensitivity

  • Apply antibody-based enrichment of dihydrouridine-containing RNAs

  • Use digital droplet PCR for quantification of specific modified tRNAs

• Controlled stress experiments:
  Design experiments with precisely controlled stress conditions:

  • Temperature stress (10°C, 25°C, 40°C)

  • Drought (controlled water limitation)

  • Salt stress (0, 50, 100, 150 mM NaCl)

  • Combined stresses to mimic field conditions

• Time-course studies:

  • Monitor changes in dihydrouridine levels at multiple timepoints after stress application

  • Correlate with transcriptomic and proteomic changes

• Genetic approaches:

  • Generate CRISPR/Cas9 mutants of individual and combined DUS genes

  • Create tissue-specific knockdowns using RNA interference

  • Develop overexpression lines to assess gain-of-function effects

• Systems biology integration:

  • Correlate dihydrouridine modification with translational efficiency

  • Model the impact of tRNA modifications on codon usage during stress

  • Integrate data across transcriptome, epitranscriptome, and proteome