AGO2 (1-200) Human

What is AGO2 (1-200) Human Recombinant protein?

AGO2 (1-200) Human Recombinant is a single, non-glycosylated polypeptide chain containing 210 amino acids (1-200 a.a.) with a molecular mass of 23.7kDa. The recombinant protein includes a 10 amino acid His-tag at the N-terminal to facilitate purification and detection . This protein represents the first 200 amino acids of the human Argonaute 2 protein, which is known by several synonyms including Protein argonaute-2, hAgo2, Argonaute RISC catalytic component 2, and Eukaryotic translation initiation factor 2C 2 .

What domains are present in full-length AGO2 and how do they compare to the truncated AGO2 (1-200)?

Full-length AGO2 comprises four distinct domains:

The AGO2 (1-200) fragment primarily contains the N-terminal domain and only a portion of the subsequent domains, lacking the complete structural elements needed for catalytic activity. This truncation is useful for studying specific protein-protein interactions involving the N-terminal region without interference from RNA slicing activities .

How does AGO2 participate in RNA interference mechanisms?

AGO2 functions as the catalytic component of the RNA-induced silencing complex (RISC). The protein binds to small non-coding RNA molecules (primarily microRNAs) that serve as "guide" sequences . This RNA-protein complex then:

• Recognizes target mRNAs through base-pairing between the guide RNA and complementary sequences

• Induces either translational repression (with partial complementarity) or mRNA cleavage (with perfect complementarity)

• Facilitates recruitment of additional protein factors for downstream effects

Recent research has expanded our understanding of AGO2's guide repertoire beyond microRNAs to include fragments of other small RNAs such as tRNAs, YRNAs, snoRNAs, and rRNAs, suggesting broader regulatory roles in cellular processes .

What tagging strategies are optimal for AGO2 studies?

Researchers should carefully consider tagging location when designing AGO2 constructs, as shown in comparative studies:

For optimal results, N-terminal tagging is recommended as it preserves most native functions of AGO2. When using HaloTag technology, N-terminal fusion generally maintains RNA silencing activity, though with some minor alterations compared to unmodified AGO2. For studying AGO2 (1-200), caution should be exercised as even N-terminal tags might affect the truncated protein's limited functional repertoire .

What is AGO2-CLASH and how can it be implemented in a research setting?

AGO2-CLASH (Cross-Linking, Ligation, And Sequencing of Hybrids) is an advanced experimental method for identifying direct AGO2 "guide"-"target" interactions. The methodology involves:

• Cross-linking: UV irradiation to create covalent bonds between AGO2 and associated RNAs

• Immunoprecipitation: Isolation of AGO2-RNA complexes

• Ligation: Joining guide RNAs and target RNAs that are in close proximity

• Library preparation: Converting isolated complexes to sequencing-ready format

• High-throughput sequencing: Generating chimeric reads that contain both guide and target sequences

• Bioinformatic analysis: Using specialized pipelines (e.g., HybriDetector) to identify genuine interactions

This technique provides a more direct visualization of AGO2 targeting than traditional CLIP approaches, generating thousands of verified AGO2 target sites that can serve as training data for computational prediction models .

How can researchers validate the functionality of AGO2 constructs?

A comprehensive validation strategy should include:

• RNA silencing assays: Measure the ability of AGO2 constructs to repress reporter genes (e.g., luciferase) through both siRNA and miRNA pathways.

• Protein interaction analysis: Assess binding to key partners like TNRC6A using co-immunoprecipitation or proximity labeling techniques.

• Subcellular localization: Examine distribution patterns, particularly nuclear/cytoplasmic ratio and P-body association, using confocal microscopy.

• RNA cleavage activity: Employ in vitro slicing assays with perfectly complementary targets to measure catalytic function.

• RNA binding analysis: Evaluate association with different RNA species through RNA immunoprecipitation followed by sequencing (RIP-seq).

These validation steps are essential for ensuring experimental results are not compromised by functional defects in the AGO2 construct, particularly when working with truncated versions like AGO2 (1-200) .

How does structural dynamics analysis inform AGO2 function?

Molecular dynamics simulations of AGO2 provide valuable insights into its functional mechanisms:

• Domain flexibility patterns: The PIWI domain of AGO2 exhibits reduced movement compared to other domains, consistent with its role as the catalytic core. Conversely, the N-terminal domain shows larger conformational alterations, suggesting potential roles in protein-protein interactions .

• Catalytic tetrad dynamics: In AGO2-AGO3, the catalytic tetrad (CT) site follows a limited mobility pattern. The first and third CT residues consistently show less flexibility than the second residue, potentially reflecting their critical roles in maintaining the active site configuration .

• Conserved interaction networks: AGO2's CT site displays a conserved interaction pattern where lysine K709 (located approximately 40 amino acids from the third CT amino acid) interacts with all tetrad residues except the second one. Additionally, the second CT residue interacts with arginine R710, which has been identified as a miRNA binding site .

These structural insights help researchers design more targeted experiments to probe AGO2 function and develop strategies for modulating its activity in research applications.

What is the AGO2 targetome and how has it been characterized?

The AGO2 targetome encompasses all RNA molecules directly bound and regulated by AGO2. Recent advances in its characterization include:

• Experimental approaches:

  • AGO2-CLASH: Generates chimeric reads containing both guide and target sequences

  • AGO2 eCLIP: Identifies AGO2 binding sites across the transcriptome

  • HEAP-seq: Uses HaloTag-AGO2 fusion for more stringent isolation of binding partners

• Key findings:

  • AGO2 associates with a diverse repertoire of small RNAs beyond canonical microRNAs

  • Guide RNAs derived from tRNAs, YRNAs, snoRNAs, and rRNAs participate in AGO2-mediated regulation

  • Different guides exhibit distinct targeting rules and preferences

• Computational modeling:

  • Convolutional neural networks trained on chimeric read data can predict binding potential for different guide classes

  • These models highlight sequence and structural features that influence AGO2 binding specificity

Understanding the complete AGO2 targetome is critical for interpreting its roles in development, disease processes, and potential therapeutic applications.

How do AGO2-protein interactions influence its regulatory functions?

AGO2 engages in multiple protein interactions that modulate its activity:

• GW182/TNRC6 interaction: AGO2 binds to Glycine-tryptophan repeat-containing protein (GW182, also known as TNRC6), controlling mRNA degradation. C-terminal tagging of AGO2 significantly reduces this interaction, highlighting the importance of the C-terminal region for protein partner recruitment .

• ZSWIM8 association: AGO2 interacts with Zinc Finger SWIM-Type Containing 8 (ZSWIM8), which is involved in target-directed miRNA degradation (TDMD). Analysis of AGO2-ZSWIM8 interaction sites shows minimal differences in molecular movements compared to other AGO proteins .

• Post-translational modifications: AGO2 contains numerous PTM sites where fluctuations range from approximately 1 to 9 Å, potentially regulating interactions with protein partners and RNA targets .

• Conserved binding interfaces: AGO2 contains long common protein subsequences (LCS1 and LCS2) located in the nucleic acid binding channel. These highly conserved regions show minimal differences in secondary protein structure and may represent critical interfaces for protein-protein interactions .

What technical challenges arise when working with AGO2 (1-200) and how can they be addressed?

Researchers working with AGO2 (1-200) encounter several technical challenges:

• Limited functionality: The truncated protein lacks complete functional domains, particularly the catalytic PIWI domain. Solution: Use AGO2 (1-200) specifically for studying N-terminal interactions rather than RNA slicing activities.

• Protein stability issues: Truncated proteins may exhibit folding problems or aggregation. Solution: Optimize buffer conditions (consider adding glycerol, reducing agents, or specific ions) and storage protocols (avoid freeze-thaw cycles).

• Tag interference: Even N-terminal tags may affect the already limited functionality of truncated AGO2. Solution: Compare multiple tagging strategies and include untagged controls wherever possible.

• Non-specific binding: Partial protein domains may expose hydrophobic regions that cause non-specific interactions. Solution: Include more stringent washing steps in pull-down experiments and appropriate negative controls.

• Verification challenges: Confirming proper folding of the truncated protein can be difficult. Solution: Employ circular dichroism or limited proteolysis to assess structural integrity.

What bioinformatic resources are available for analyzing AGO2 interactions?

Several specialized tools have been developed for AGO2 research:

These computational resources enable researchers to analyze AGO2-RNA interactions, predict binding sites, and compare structural features across experimental conditions .

How can researchers distinguish between specific and non-specific AGO2-RNA interactions?

Differentiating genuine AGO2-RNA interactions from background noise requires a multi-faceted approach:

• Experimental strategies:

  • Employ covalent tagging systems (like HaloTag) that allow stringent washing conditions

  • Use chimeric read analysis methods that provide direct evidence of guide-target proximity

  • Include appropriate negative controls (AGO2 knockout, non-relevant RNA pulldowns)

• Data filtering approaches:

  • Implement computational models trained on verified interactions to score potential binding sites

  • Apply stringent statistical thresholds that account for background binding levels

  • Compare binding profiles across multiple experimental replicates

• Validation methods:

  • Confirm predicted interactions using reporter gene assays

  • Perform direct binding measurements with purified components

  • Analyze binding site characteristics for expected features (e.g., seed complementarity)

By combining rigorous experimental design with sophisticated computational analysis, researchers can build high-confidence datasets of AGO2-RNA interactions that advance our understanding of its regulatory networks .

What are emerging applications for AGO2 (1-200) in RNA biology research?

Recent advances suggest several promising research directions:

• Structural biology applications: The N-terminal fragment can serve as a model system for understanding domain-specific interactions without the complexity of the full protein.

• Interaction screening: AGO2 (1-200) can be used in high-throughput screens to identify novel protein partners that specifically interact with the N-terminal region.

• Domain-specific inhibitor development: The truncated protein provides a platform for developing compounds that selectively target N-terminal interactions without affecting catalytic activity.

• Comparative studies: Parallel analysis of AGO2 (1-200) alongside other AGO protein fragments can illuminate evolutionary relationships and functional specialization.

These applications leverage the simplified structure of AGO2 (1-200) to address specific questions about domain function and protein-protein interactions in RNA regulatory networks .

How do contradictory findings about AGO2 function inform experimental design?

The literature contains several apparently contradictory findings about AGO2 function, which researchers should consider when designing experiments:

• Tagging effects: While N-terminal tagging generally preserves AGO2 function, some studies report altered activities even with N-terminal tags. Solution: Include untagged controls and validate key findings with multiple tagging strategies.

• Cell type specificity: AGO2 behavior varies across cell types, potentially due to different expression levels of cofactors. Solution: Verify findings across multiple relevant cell types.

• Guide RNA diversity: The expanding repertoire of AGO2-associated guide RNAs (beyond microRNAs) may explain seemingly contradictory results when different RNA populations are enriched. Solution: Characterize the complete guide RNA profile in your specific experimental system.

• Post-translational modifications: Variable PTM patterns can dramatically alter AGO2 function. Solution: Consider the PTM status of AGO2 in your system and how experimental conditions might affect modifications.

These considerations highlight the importance of comprehensive validation and careful interpretation of results in AGO2 research .