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Related Experiment Video

Updated: Sep 14, 2025

Aip1p Dynamics Are Altered by the R256H Mutation in Actin

Aip1p Dynamics Are Altered by the R256H Mutation in Actin

Published on: July 30, 2014

C-terminal tagging impairs AGO2 function.

Kunal M Shah¹, Alex F F Crozier¹, Anika Assaraf^1,2

¹Centre for Cancer Cell and Molecular Biology, Barts Cancer Institute, Queen Mary University of London, London, UK.

|July 23, 2025

Summary

This summary is machine-generated.

C-terminal HaloTag fusions of Argonaute 2 (AGO2) impair its gene silencing function and localization. This study highlights the need to validate tagging strategies for accurate RNA silencing research.

Keywords:

AGO2 Argonaute CRISPR-Cas9 HaloTag RNAi microRNAs protein tagging

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Published on: June 30, 2022

Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

MicroRNA-mediated gene silencing is crucial for post-transcriptional gene regulation.
Argonaute 2 (AGO2) is central to the RNA-induced silencing complex (RISC).
Studying AGO2 function requires reliable molecular tools, but tagging can introduce artifacts.

Purpose of the Study:

To investigate the functional consequences of C-terminal HaloTagging of AGO2.
To compare the effects of N-terminal versus C-terminal tagging on AGO2 function.
To assess the suitability of C-terminal HaloTag-AGO2 for studying RISC biology.

Main Methods:

Generated a C-terminal HaloTag fusion of AGO2 (AGO2HALO) using CRISPR-Cas9 (CRISPaint).
Assessed AGO2HALO protein binding, cell viability, RNA cleavage, silencing activity, and localization.
Compared N-terminal (AGO2-EGFP) and C-terminal (EGFP-AGO2) tagged AGO2 via transient transfection.

Main Results:

C-terminal AGO2HALO showed reduced TNRC6A binding, impaired RNA cleavage, silencing, and nuclear localization.
N-terminal AGO2-EGFP retained wild-type-like function and localization.
C-terminal EGFP-AGO2 was impaired in silencing, nuclear import, and P-body localization.

Conclusions:

C-terminal HaloTagging compromises AGO2 functionality, making it unsuitable for RISC studies.
Tagging strategies must be validated to prevent artifact-induced conclusions in gene silencing research.
N-terminal tagging appears more compatible with maintaining AGO2 function compared to C-terminal tagging.