Shah Kunal M, Crozier Alex F F, Assaraf Anika, Arya Muzjda, Grevitt Paul, Mardakheh Faraz, Plevin Michael J, Sharp Tyson V
Centre for Cancer Cell and Molecular Biology, Barts Cancer Institute, Queen Mary University of London, London, UK.
University Hospital Southampton NHS Foundation Trust - Southampton Biomedical Research Centre, Southampton.
RNA Biol. 2025 Dec;22(1):1-24. doi: 10.1080/15476286.2025.2534028. Epub 2025 Jul 23.
MicroRNA-mediated gene silencing is a conserved mechanism of post-transcriptional gene regulation across metazoans. It depends on base pairing between small RNAs and mRNAs, and on protein complexes including the RNA-induced silencing complex (RISC), where Argonaute 2 (AGO2) plays a central role. A full understanding of RNA silencing requires reliable molecular tools to study AGO2 and RISC. Affinity tagging and antibody-based methods can introduce artefacts, and both the - and C-terminal domains of AGO2 are critical for its function. While N-terminal tags are frequently used, and a recent study in mice showed altered activity in N-terminal HaloTag-AGO2 fusions, the consequences of C-terminal tagging remain underexplored. CRISPaint, a CRISPR-Cas9-based technique, enables endogenous C-terminal tag fusions without requiring homology arms. Using this system, we generated the first C-terminal HaloTag fusion of AGO2 (AGO2HALO) in human A549 cells. We found that the AGO2HALO fusion protein exhibits reduced binding with TNRC6A, with no effect on cell viability. However, it significantly impairs RNA cleavage, silencing activity, and nuclear localization. We further compared AGO2-EGFP and EGFP-AGO2 using transient transfection. N-terminally tagged AGO2 retained wild-type-like function and localization, while C-terminally tagged AGO2 was impaired in siRNA and miRNA silencing, nuclear import, and P-body localization. These results demonstrate that a C-terminal HaloTag compromises AGO2 functionality and is unsuitable for studying RISC biology. Our findings highlight the importance of validating tagging strategies to avoid misleading conclusions due to tag-induced functional defects. Pre-print, bioRxiv.
微小RNA介导的基因沉默是后生动物中一种保守的转录后基因调控机制。它依赖于小RNA与mRNA之间的碱基配对，以及包括RNA诱导沉默复合体（RISC）在内的蛋白质复合体，其中AGO2（Argonaute 2）起着核心作用。要全面了解RNA沉默，需要可靠的分子工具来研究AGO2和RISC。亲和标签法和基于抗体的方法可能会引入人为因素，并且AGO2的N端和C端结构域对其功能都至关重要。虽然N端标签经常被使用，最近一项对小鼠的研究表明N端HaloTag-AGO2融合蛋白的活性发生了改变，但C端标签的影响仍未得到充分探索。CRISPaint是一种基于CRISPR-Cas9的技术，无需同源臂即可实现内源性C端标签融合。利用该系统，我们在人A549细胞中生成了首个AGO2的C端HaloTag融合蛋白（AGO2HALO）。我们发现AGO2HALO融合蛋白与TNRC6A的结合减少，对细胞活力没有影响。然而，它显著损害了RNA切割、沉默活性和核定位。我们通过瞬时转染进一步比较了AGO2-EGFP和EGFP-AGO2。N端标记的AGO2保留了野生型样的功能和定位，而C端标记的AGO2在siRNA和miRNA沉默、核输入和P小体定位方面受损。这些结果表明，C端HaloTag会损害AGO2的功能，不适用于研究RISC生物学。我们的发现强调了验证标签策略的重要性，以避免因标签诱导的功能缺陷而得出误导性结论。预印本，bioRxiv。
