Recombinant Arabidopsis thaliana Pentatricopeptide repeat-containing protein At2g40720 (PCMP-E26), partial

What are Pentatricopeptide Repeat (PPR) proteins in Arabidopsis thaliana?

PPR proteins in Arabidopsis thaliana represent one of the largest and most enigmatic gene families discovered through genome sequencing. They are characterized by tandem arrays of pentatricopeptide repeats and play constitutive, often essential roles in mitochondria and chloroplasts. According to comprehensive bioinformatic analysis, there are 441 members of the Arabidopsis PPR family .

The primary function of these proteins involves binding to organellar transcripts, which has been confirmed through detailed characterization of individual mutants and RNA binding assays. Research has shown that many PPR genes have essential functions in plant embryos, and mutations in these genes can lead to embryo abortion .

When studying a specific PPR protein like At2g40720 (PCMP-E26), researchers should analyze its subcellular localization, RNA targets, and potential role in RNA editing, stability, or processing based on the general understanding of PPR protein functions.

What experimental approaches are most effective for determining the subcellular localization of At2g40720?

PPR proteins in Arabidopsis are predominantly localized in mitochondria and chloroplasts. According to research, this localization has been confirmed through GFP and RFP fusion studies . For determining the localization of At2g40720, researchers should:

• Perform in silico analysis using prediction algorithms (TargetP, Predotar) to identify potential transit peptides

• Generate fluorescent protein fusions (GFP/RFP) with the following components:

  • Full-length At2g40720 coding sequence

  • N-terminal region (first 100 amino acids) only

  • C-terminal fusion versus N-terminal fusion constructs to assess potential targeting disruption

• Express these constructs in Arabidopsis protoplasts for rapid assessment

• Create stable transgenic lines under native or constitutive promoters

• Image using confocal microscopy and co-localize with organelle-specific markers

This methodological approach provides empirical evidence for subcellular localization, which is crucial for understanding the function of At2g40720 in the context of organellar gene expression.

What are the predicted RNA targets of At2g40720 based on its PPR motif structure?

While the search results don't provide specific information about At2g40720's RNA targets, a methodological approach to predict targets includes:

• Motif analysis:

  • Identify the specific PPR motifs present in At2g40720

  • Map the amino acids at positions 6 and 1' in each PPR motif, which are critical for nucleotide recognition

  • Apply the PPR code to predict RNA sequence recognition

• Comparative analysis:

  • Identify closest homologs with known RNA targets

  • Compare PPR motif patterns to infer potential similar target sequences

• Validation methodology:

  • Generate recombinant At2g40720 protein using seed storage protein 3'UTR for high expression in Arabidopsis

  • Perform RNA immunoprecipitation followed by sequencing (RIP-seq)

  • Conduct in vitro binding assays with predicted target RNA sequences

This structured approach helps establish potential RNA targets before committing to more extensive experimental validation studies.

How can researchers optimize expression of recombinant At2g40720 for functional studies?

Expressing recombinant PPR proteins presents unique challenges due to their repetitive structure and specific subcellular targeting. Based on recent research, the following methodological approach is recommended:

For expression in Arabidopsis:

• Utilize the 3'UTR of seed storage protein (SSP) genes fused to the At2g40720 coding sequence, as this approach has been shown to lead to massive accumulation of recombinant proteins with preserved enzymatic activity in Arabidopsis seeds .

• Design a construct that includes:

  • A strong seed-specific promoter (e.g., β-phaseolin)

  • The At2g40720 coding sequence

  • The 3'UTR of an SSP gene

The use of 3'UTR from SSP genes is particularly effective because "SSPs comprise the major proportion of proteins in seeds" and the 3'UTR elements are "essential for SSP accumulation" . This approach provides a cost-effective method for obtaining functional At2g40720 protein.

What are the challenges in identifying RNA editing sites regulated by At2g40720?

Identifying RNA editing sites regulated by specific PPR proteins like At2g40720 requires a systematic approach to overcome several challenges:

• Methodological workflow for RNA editing site identification:

  • Generate At2g40720 knockout or knockdown lines (T-DNA insertion, CRISPR-Cas9, RNAi)

  • Extract RNA from both wild-type and mutant plants

  • Perform strand-specific RNA sequencing of organellar transcripts

  • Conduct comparative analysis to identify C-to-U editing changes between genotypes

  • Validate candidate sites using targeted RT-PCR and Sanger sequencing

• Potential challenges and solutions:

• Validation approaches:

  • Complementation studies with wild-type At2g40720

  • In vitro editing assays with recombinant protein

  • Direct binding studies with identified target RNAs

This comprehensive approach addresses the complexity of identifying RNA editing sites in organellar transcripts regulated by specific PPR proteins.

How do mutations in At2g40720 affect the Arabidopsis transcriptome and metabolome?

The analysis of transcriptomic and metabolomic changes in At2g40720 mutants requires sophisticated experimental design:

• Experimental design for multi-omics analysis:

  • Generate at least three independent mutant lines (complete knockout, partial loss-of-function)

  • Grow plants under controlled conditions with biological replicates

  • Sample at multiple developmental stages and/or stress conditions

  • Perform parallel RNA-seq, proteomics, and metabolomics analyses

• Transcriptome analysis methodology:

  • Strand-specific RNA-seq of both nuclear and organellar transcripts

  • Analysis of differential gene expression, alternative splicing, and RNA editing

  • Pathway enrichment analysis for affected genes

  • Special attention to organellar transcripts most likely to be direct targets

• Metabolome analysis methodology:

  • Targeted analysis of primary metabolites using GC-MS

  • Untargeted metabolomics using LC-MS

  • Focus on metabolic pathways dependent on organellar function

• Integration of multi-omics data:

  • Correlation analysis between transcriptome and metabolome changes

  • Network analysis to identify key perturbed pathways

  • Comparison with known PPR mutant phenotypes

As research has shown that "many PPR genes have an essential function in plant embryos" , researchers should consider using inducible or tissue-specific mutations if complete knockout causes lethality.

What are the key considerations for designing experiments with recombinant At2g40720?

When designing experiments involving recombinant At2g40720, several methodological considerations should be addressed:

• Construct design:

  • Include appropriate promoters (tissue-specific or constitutive depending on the research question)

  • Consider fusion of seed storage protein 3'UTR for enhanced expression in seeds

  • Add epitope tags (His, FLAG, HA) for purification and detection

  • Include flexible linkers between the protein and tags to maintain function

  • Ensure the N-terminal targeting sequence remains intact if organellar localization is desired

• Expression system selection:

  • For biochemical characterization: heterologous expression in E. coli or insect cells

  • For in planta function: stable transformation of Arabidopsis (preferably in the corresponding mutant background)

  • For localization studies: transient expression in Arabidopsis protoplasts

• Regulatory compliance:

  • Ensure compliance with NIH Guidelines for recombinant DNA research

  • Note that "experiments covered by both III-D or III-E and III-F are considered to be exempt" from the NIH Guidelines

  • Be aware that while "nucleic acid molecules resulting from the replication of recombinant or synthetic nucleic acids are subject to the NIH Guidelines," the proteins produced from them are not

• Validation experiments:

  • Confirm protein expression by Western blot

  • Verify subcellular localization using microscopy

  • Test RNA binding capacity with electrophoretic mobility shift assays

  • Assess functional complementation in mutant backgrounds

These methodological considerations ensure robust experimental design and reliable results when working with recombinant At2g40720.

How can RNA-protein interactions be quantitatively assessed for At2g40720?

Quantitative assessment of RNA-protein interactions for At2g40720 requires multiple complementary approaches:

• In vitro binding assays:

• In vivo interaction analysis:

  • RNA immunoprecipitation followed by sequencing (RIP-seq)

  • Cross-linking immunoprecipitation (CLIP-seq)

  • Proximity-dependent biotin identification (BioID) coupled with RNA isolation

• Validation methodology:

  • Compare binding to mutated RNA sequences to define specificity determinants

  • Analyze RNA secondary structure effects on binding

  • Competition assays with other PPR proteins targeting similar sequences

• Data analysis considerations:

  • Fit binding data to appropriate models (single-site binding, cooperative binding)

  • Calculate confidence intervals for binding constants

  • Compare affinity across different RNA targets

These methodological approaches provide quantitative measurements of RNA-protein interactions, which are essential for understanding the molecular function of At2g40720.

What are the best approaches for analyzing the structure-function relationship of At2g40720?

Analyzing the structure-function relationship of At2g40720 requires a comprehensive approach combining computational and experimental methods:

This integrated approach allows researchers to correlate structural features of At2g40720 with its functional properties, providing insights into the molecular mechanism of action.

What NIH Guidelines apply to research with recombinant At2g40720?

Research involving recombinant At2g40720 must comply with NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. Based on the search results, the following methodological guidance applies:

• Exempt status determination:

  • Research may be exempt if it falls under Section III-F of the NIH Guidelines

  • Specifically, experiments that "consist entirely of nucleic acids from a eukaryotic host including its chloroplasts, mitochondria, or plasmids (but excluding viruses) when propagated only in that host (or a closely related strain of the same species)" may be exempt

  • Since At2g40720 is native to Arabidopsis, research involving its recombinant expression in Arabidopsis would likely fall under this exemption

• Non-exempt research considerations:

  • Research would not be exempt if it involves:

    • DNA from Risk Group 3 or 4 organisms

    • Whole plants regenerated from plant cells that do not remain axenic cultures

    • Large scale experiments (more than 10 liters of culture volume)

• Important regulatory notes:

  • "If an experiment falls into Section III-D or III-E of the NIH Guidelines and also falls into section III-F, it is exempt"

  • While "nucleic acid molecules resulting from the replication of recombinant or synthetic nucleic acids are subject to the NIH Guidelines," the proteins produced by genetically engineered organisms are not

• Practical implementation steps:

  • Consult with institutional biosafety committee (IBC)

  • Document risk assessment

  • Obtain IBC approval before beginning non-exempt research

  • Maintain records of all approvals and modifications

This regulatory framework ensures proper oversight while allowing scientific progress in understanding PPR proteins like At2g40720.

What controls and experimental validation are essential for studies on At2g40720 function?

Rigorous experimental validation of At2g40720 function requires appropriate controls and validation steps:

• Essential controls for gene function studies:

• Methodological validation steps:

  • Verify gene knockout/knockdown by RT-PCR and Western blot

  • Confirm protein expression and purity for biochemical studies

  • Validate antibody specificity using knockout lines as negative controls

  • Sequence verification of all constructs before functional studies

  • Biological replicates (minimum n=3) for all quantitative analyses

• Data validation approaches:

  • Statistical analysis appropriate to experimental design

  • Multiple independent methods to confirm key findings

  • Dose-response experiments where applicable

  • Temporal analysis to distinguish direct from indirect effects

These methodological controls and validation steps ensure reproducible and reliable results when studying At2g40720 function, addressing potential sources of experimental artifacts or misinterpretation.

How can CRISPR-Cas technologies be applied to study At2g40720 function?

CRISPR-Cas technologies offer powerful approaches for studying At2g40720 function:

• Gene editing strategies:

  • Complete gene knockout using paired sgRNAs to delete the entire locus

  • Targeted mutagenesis of specific PPR motifs to alter RNA binding specificity

  • Introduction of point mutations to test structure-function hypotheses

  • C-terminal tagging with fluorescent proteins or epitope tags at the endogenous locus

• Transcriptional modulation:

  • CRISPRi (dCas9-KRAB) for transcriptional repression

  • CRISPRa (dCas9-VP64) for transcriptional activation

  • Inducible systems for temporal control of expression

• RNA targeting approaches:

  • Cas13-based targeting of At2g40720 mRNA

  • Visualization of At2g40720-RNA interactions using CRISPR-based imaging

• Methodological considerations:

  • Design of highly specific sgRNAs to minimize off-target effects

  • Efficient delivery methods for Arabidopsis transformation

  • Screening strategies for identifying edited plants

  • Genetic complementation to confirm phenotypes

These CRISPR-based approaches enable precise manipulation of At2g40720 in its endogenous context, providing insights into its function that would be difficult to obtain through traditional methods.

What is the current understanding of the evolution of PPR proteins like At2g40720 in plants?

While the search results don't provide specific information about the evolution of At2g40720, a methodological approach to studying PPR protein evolution includes:

• Phylogenetic analysis methodology:

  • Identify At2g40720 orthologs across plant species

  • Construct multiple sequence alignments of PPR domains

  • Generate phylogenetic trees using maximum likelihood or Bayesian approaches

  • Map the emergence of specific functional domains or motifs

• Comparative genomics approaches:

  • Analyze synteny around At2g40720 loci across species

  • Examine patterns of gene duplication and diversification

  • Compare selection pressure (dN/dS ratios) on different PPR family members

  • Analyze co-evolution with organellar RNA targets

• Functional evolution studies:

  • Test complementation of Arabidopsis At2g40720 mutants with orthologs from other species

  • Compare RNA binding specificities of orthologous proteins

  • Analyze correlated changes in editing sites and PPR proteins across species

Understanding the evolutionary history of At2g40720 provides context for its current function and helps predict its role in different plant species.