Recombinant Arabidopsis thaliana Pentatricopeptide repeat-containing protein At1g55630 (At1g55630)

What is At1g55630 and how is it classified within the PPR protein family?

At1g55630 is a pentatricopeptide repeat-containing protein found in Arabidopsis thaliana. It belongs to the larger family of PPR proteins, which are characterized by tandem arrays of a degenerate 35-amino-acid repeat motif. The Arabidopsis genome contains 458 annotated PPR genes, making it one of the largest protein families in plants . At1g55630 specifically contains the PPR motif that enables RNA binding through specific amino acid patterns that recognize RNA bases, similar to how TAL effector proteins function .

To determine the specific subclass of At1g55630:

• Analyze the protein sequence using PPR motif recognition tools

• Compare with known PPR protein classifications (P, PLS, E, E+, or DYW subfamilies)

• Identify additional domains that may be present outside the PPR repeats

What is the predicted structure of At1g55630 and how does it relate to its function?

The structure of At1g55630 follows the characteristic PPR protein architecture featuring:

• N-terminal targeting sequence (for organellar localization)

• Multiple PPR motifs arranged in tandem

• Each PPR motif adopts a helix-turn-helix structure forming a solenoid

• Specific amino acids at positions 6 and 1' in each repeat are critical for RNA base recognition

The structure-function relationship can be analyzed by:

• Homology modeling based on solved PPR protein structures

• Predicting RNA binding sites using the established PPR code

• Site-directed mutagenesis of key residues to validate structural predictions

• Circular dichroism spectroscopy to assess secondary structure composition

What is the predicted subcellular localization of At1g55630?

Based on the general characteristics of PPR proteins, At1g55630 is likely targeted to either mitochondria, chloroplasts, or potentially both organelles. Prediction algorithms and experimental data should be consulted to determine its precise localization .

For experimental verification of localization:

• Express At1g55630 fused to a fluorescent protein (GFP or RFP)

• Transform plant cells (protoplasts) and observe using confocal microscopy

• Co-localize with known organelle markers

• Confirm with subcellular fractionation and immunoblotting

The systematic study of PPR protein localization by Lurin et al. revealed that many PPR proteins with ambiguous targeting predictions were experimentally confirmed to localize to organelles, with some showing dual targeting to both mitochondria and chloroplasts .

How can we experimentally determine if At1g55630 is dual-targeted to both mitochondria and chloroplasts?

To determine if At1g55630 shows dual targeting to both organelles:

• Targeting peptide analysis:

  • Clone the N-terminal region (first 100-150 amino acids) of At1g55630

  • Fuse to a reporter protein (RFP)

  • Transform into plant protoplasts

  • Observe localization using confocal microscopy with organelle-specific markers

• Full-length protein localization:

  • Express the complete At1g55630 protein fused to a fluorescent tag

  • Compare results with the targeting peptide experiment

  • Look for signals in both organelles

• Biochemical confirmation:

  • Perform subcellular fractionation to isolate pure mitochondria and chloroplasts

  • Use western blotting with anti-At1g55630 antibodies to detect the protein in both fractions

  • Include controls for organelle-specific markers to confirm fraction purity

Lurin et al. developed a standardized methodology for PPR protein localization, which revealed that many PPR proteins with ambiguous targeting predictions showed experimentally confirmed dual localization .

What RNA targets does At1g55630 potentially interact with?

While specific RNA targets for At1g55630 have not been definitively identified in the provided search results, methodological approaches to determine its targets include:

• Computational prediction:

  • Analyze the PPR motifs in At1g55630 using the PPR-RNA recognition code

  • Scan organellar transcriptomes for potential binding sites

  • Compare with targets of closely related PPR proteins

• RNA immunoprecipitation (RIP):

  • Express tagged At1g55630 in plants

  • Immunoprecipitate the protein-RNA complexes

  • Identify bound RNAs through sequencing (RIP-seq)

• Crosslinking and immunoprecipitation (CLIP):

  • Use UV crosslinking to capture direct RNA-protein interactions

  • Immunoprecipitate At1g55630

  • Sequence associated RNAs

• Artificial PPR approach:

  • Design synthetic PPR proteins based on At1g55630's binding specificity

  • Use them as RNA affinity tags to isolate ribonucleoprotein complexes

  • This approach has been successfully used for other PPR proteins

How can we determine the specific function of At1g55630 in post-transcriptional regulation?

To elucidate the specific function of At1g55630 in post-transcriptional regulation:

• Loss-of-function analysis:

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

  • Analyze changes in RNA processing, including:

    • RNA stability (half-life measurements)

    • RNA editing sites (sequence comparisons between mutant and wild-type)

    • Splicing patterns (RT-PCR across introns)

    • Translation efficiency (polysome profiling)

• Gain-of-function analysis:

  • Overexpress At1g55630 and analyze effects on target RNAs

  • Express in heterologous systems to assess specific RNA processing activities

• In vitro RNA binding and processing assays:

  • Express and purify recombinant At1g55630

  • Perform electrophoretic mobility shift assays (EMSA)

  • Test RNA editing, stability, or other processing activities in vitro

PPR proteins commonly function in RNA stabilization, splicing, editing, and translation, making these processes primary candidates for investigation .

What are the best methods for producing recombinant At1g55630 protein for in vitro studies?

For producing high-quality recombinant At1g55630:

• Expression system selection:

  • E. coli: Most common, but may have issues with solubility of plant proteins

    • Use specialized strains (Rosetta, Arctic Express) for rare codons and folding

    • Consider fusion tags (MBP, SUMO) to enhance solubility

  • Insect cells: Better for eukaryotic proteins requiring post-translational modifications

  • Plant-based expression: Consider for authentic folding and modifications

• Construct design considerations:

  • Remove the organelle targeting peptide (first ~50-100 amino acids)

  • Add appropriate purification tags (His, GST, FLAG)

  • Consider codon optimization for the expression host

• Purification strategy:

  • Affinity chromatography based on fusion tag

  • Ion exchange chromatography

  • Size exclusion chromatography for final polishing

  • Verify protein quality by SDS-PAGE and mass spectrometry

• Activity assessment:

  • RNA binding assays (EMSA, filter binding)

  • Structural analysis (circular dichroism, thermal shift)

The antibody product information (CSB-PA773700XA01DOA) indicates that recombinant At1g55630 has been successfully produced for immunization purposes, suggesting feasible expression strategies exist .

What antibody resources are available for At1g55630 and how can they be validated?

Based on the search results, at least one commercial antibody against At1g55630 is available:

Available resource:

• Product Code: CSB-PA773700XA01DOA

• Type: Polyclonal antibody

• Host: Rabbit

• Immunogen: Recombinant Arabidopsis thaliana At1g55630 protein

• Applications: ELISA, Western blot

• Storage: -20°C or -80°C in 50% glycerol, 0.01M PBS (pH 7.4), 0.03% Proclin 300

Validation protocol:

• Western blot analysis:

  • Include positive control (recombinant At1g55630)

  • Include negative control (extract from knockout mutant)

  • Test specificity across different plant tissues

  • Verify expected molecular weight

• Immunoprecipitation validation:

  • Perform IP followed by mass spectrometry

  • Verify pull-down of At1g55630 and associated proteins

  • Compare results with IgG control

• Immunolocalization:

  • Test antibody in immunofluorescence assays

  • Verify localization pattern matches GFP fusion results

  • Include appropriate controls

• Cross-reactivity assessment:

  • Test against closely related PPR proteins

  • Evaluate specificity across different plant species if needed

How can we design artificial PPR proteins based on At1g55630 for RNA targeting applications?

Designing artificial PPR proteins based on At1g55630:

• Decoding the RNA recognition pattern:

  • Analyze the specific amino acids at positions 6 and 1' in each PPR repeat

  • Map these to the corresponding RNA bases according to the established PPR code

  • Determine At1g55630's native RNA binding specificity

• Engineering modified binding specificity:

  • Design custom PPR repeat arrays by modifying the amino acids at positions 6 and 1'

  • Use computational modeling to predict binding to target RNA sequences

  • Create synthetic PPR proteins with novel specificities

• Construct design:

  • Maintain the structural scaffold of At1g55630

  • Replace or modify specific repeats to alter binding specificity

  • Add functional domains for specific applications (e.g., RNA editing, cleavage)

• Validation strategies:

  • In vitro binding assays to confirm target specificity

  • Cellular assays to verify function in vivo

  • Pull-down experiments to identify bound RNAs and associated proteins

This approach has been successfully implemented for other PPR proteins, as demonstrated by McDermott et al. who designed artificial PPRs to specifically bind chloroplast psbA mRNA for ribonucleoprotein particle isolation .

How does At1g55630 compare to other PPR proteins in Arabidopsis thaliana in terms of sequence conservation and functional divergence?

Comprehensive comparative analysis of At1g55630:

• Phylogenetic analysis:

  • Align At1g55630 with all 458 PPR proteins in Arabidopsis

  • Generate phylogenetic trees to identify closest relatives

  • Determine evolutionary relationships and potential functional clusters

• Domain architecture comparison:

  • Analyze the number and arrangement of PPR repeats

  • Identify additional functional domains

  • Compare N-terminal targeting sequences

• Expression pattern analysis:

  • Compare tissue-specific expression patterns

  • Analyze expression under various stress conditions

  • Identify co-expressed genes

• Functional comparison:

  • Contrast RNA targets if known

  • Compare subcellular localization data

  • Analyze mutant phenotypes

• Conservation across species:

  • Identify orthologs in other plant species

  • Assess sequence conservation at key functional residues

  • Evaluate evolutionary rate compared to other PPR proteins

What is the optimal experimental design for characterizing the impact of At1g55630 knockout on plant phenotype and molecular function?

A comprehensive experimental design for At1g55630 functional characterization:

• Genetic material preparation:

  • Obtain or generate T-DNA insertion lines disrupting At1g55630

  • Create CRISPR/Cas9 knockout lines (for redundancy or as alternatives)

  • Develop complementation lines expressing At1g55630 in the knockout background

  • Create overexpression lines in wild-type background

• Phenotypic characterization:

  • Morphological analysis:

    • Plant growth measurements (height, leaf size, etc.)

    • Developmental timing assessment

    • Reproductive success evaluation

  • Physiological characterization:

    • Photosynthetic parameters

    • Respiration rates

    • Stress response (abiotic and biotic)

• Molecular characterization:

  • Transcriptome analysis:

    • RNA-seq of mutant vs. wild-type

    • Focus on organellar transcripts

    • Analyze RNA processing events (editing, splicing)

  • Organellar function:

    • Mitochondrial/chloroplast protein composition

    • Organelle morphology and ultrastructure

    • Organellar genome stability

• Biochemical characterization:

  • Identify RNA targets using RIP-seq

  • Characterize protein interaction partners via co-IP/MS

  • Assess impacts on translation using ribosome profiling

• Data integration and analysis:

  • Correlate molecular changes with phenotypic outcomes

  • Compare with known functions of related PPR proteins

  • Develop hypotheses for specific mechanistic roles

This experimental design reflects the approach used in characterizing other PPR proteins, where genetic manipulation followed by detailed phenotypic and molecular analysis has revealed functions in RNA editing, splicing, and stability .

How can contradictory data regarding At1g55630 subcellular localization be reconciled and validated?

When faced with contradictory localization data for At1g55630:

• Systematic evaluation of previous methods:

  • Review the methodologies used in contradictory studies

  • Assess potential limitations of each approach

  • Evaluate the quality controls employed

• Multi-method verification strategy:

  • In vivo fluorescent protein fusions:

    • Test both N- and C-terminal fusions

    • Use both targeting peptide and full-length protein constructs

    • Employ transient and stable transformation

  • Biochemical fractionation:

    • Use high-purity organelle isolation techniques

    • Perform western blotting with specific antibodies

    • Include markers for each subcellular compartment

  • Immunogold electron microscopy:

    • Provide high-resolution localization data

    • Quantify gold particle distribution across compartments

    • Perform statistical analysis of particle distribution

• Targeting sequence analysis:

  • Use multiple prediction algorithms (TargetP, Predotar, etc.)

  • Compare with experimentally verified dual-targeted PPR proteins

  • Analyze potential alternative translation start sites

• Developmental and stress-dependent localization:

  • Examine localization under different conditions

  • Assess tissue-specific patterns

  • Consider dynamic relocalization possibilities

The systematic study by Lurin et al. demonstrated that prediction algorithms sometimes fail to correctly identify dual-targeted PPR proteins, highlighting the importance of experimental verification .

What methodological approaches can resolve RNA targets and binding specificity of At1g55630?

To definitively identify RNA targets and characterize binding specificity:

• High-throughput in vivo approaches:

  • CLIP-seq (Crosslinking and Immunoprecipitation):

    • UV-crosslink RNA-protein complexes in vivo

    • Immunoprecipitate At1g55630

    • Sequence associated RNA fragments

    • Identify binding motifs through computational analysis

  • RIP-seq (RNA Immunoprecipitation):

    • Pull down At1g55630 under native conditions

    • Sequence associated RNAs

    • Compare with control immunoprecipitations

• In vitro binding studies:

  • SELEX (Systematic Evolution of Ligands by Exponential Enrichment):

    • Expose recombinant At1g55630 to RNA libraries

    • Select and amplify bound sequences

    • Identify preferred binding motifs after multiple rounds

  • RNA Bind-n-Seq:

    • Incubate protein with random RNA sequences

    • Sequence bound RNAs

    • Computationally derive binding motifs

  • Quantitative binding assays:

    • Determine binding affinities for predicted targets

    • Compare binding to mutated target sequences

    • Establish structure-function relationships

• Functional validation:

  • In vivo reporter systems:

    • Fuse candidate target sequences to reporter genes

    • Co-express with At1g55630 or mutant versions

    • Quantify effects on RNA stability or translation

  • Organellar run-on transcription assays:

    • Assess impact of At1g55630 on transcript stability

    • Measure processing of specific RNAs

• Integrative data analysis:

  • Cross-reference results from multiple methods

  • Validate top candidates with targeted approaches

  • Compare with binding patterns of related PPR proteins

This comprehensive approach combines the methodological power of genome-wide studies with the resolution of targeted biochemical assays, as has been successful for characterizing other PPR proteins .