A C-terminal HaloTag Impairs AGO2 Function

Abstract

AbstractA full understanding of RNA silencing requires appropriate molecular biology tools to explore the roles of Argonaute 2 (AGO2) and the RNA-induced silencing complex (RISC). Commonly used approaches to study RNA silencing and RISC, such as those relying on affinity tagging and antibodies, have important limitations that can lead to artificial results. Both the N– and C-terminal domains of AGO2 have been shown to be important for correct activity and yet the consequences of appending tags to either terminus have not been fully investigated. N-terminal tags are frequently used to study AGO(2) biology. Recently, an N-terminalHaloTag-Ago2fusion was reported and examined in mice. While the versatile HaloTag provided new opportunities to study RISC biology, the tagged construct showed certain activity changes compared to unmodified Ago2. CRISPaint, a new CRISPR-Cas9 technique, offers a route to the accurate and efficient generation of endogenous C-terminal tag fusions. Here, we used CRISPaint to generate the first reported recombinant AGO2 construct with a C-terminal tag: an endogenous C-terminal HaloTag fusion to AGO2 (AGO2HALO) in human (A549) cells. We found that the AGO2HALOfusion protein retains the capacity to interact with the key protein binding partner TNRC6A and that the C-terminal HaloTag does not affect cell viability. However, theAGO2HALOfusion significantly impairs RNA cleavage and RNA silencing activity compared to control cells, and reduces nuclear localisation of the fusion protein. We conclude that the fusion of a C-terminal HaloTag to AGO2 is not appropriate for studying AGO2 and RISC. Our results stress the importance of fully validating recombinant tagging strategies to ensure that any results generated do not obscure critical functional defects.