Crozier, A. F. F. | Search bioRxiv

bioRxiv2025

An AGO2 adaptor expands the functional and evolutionary reach of microRNA targeting

Current models of microRNA (miRNA) silencing posit that RNA-sequence rules are sufficient for canonical targeting of mRNAs by Argonaute 2 (AGO2), the central protein of the miRNA-induced silencing complex (miRISC). Using chimeric eCLIP in CRISPR-edited LIMD1+/+, LIMD1+/-, and LIMD1-/- human small airway epithelial cells (hSAECs), we reveal a transcriptome-wide dependency on LIMD1, an AGO2 adaptor, for effective miRNA targeting and repression. In LIMD1-deficient cells, miRNA loading is uncoupled from productive targeting: despite increased AGO2-miRNA interactions, complexes engage fewer transcripts and sites, reducing occupancy and more than halving both the breadth and depth of targeting. We also observe altered AGO2 positional footprints across targets in LIMD1-deficient cells. LIMD1 dependence is most pronounced at defined RNA contexts: weak (GC-poor) seed pairings, interactions involving evolutionarily young miRNAs or sites that nonetheless form thermodynamically stable duplexes, with these losses particularly enriched in coding sequences of rapidly evolving C2H2-zinc-finger genes. Even within canonical seed repertoires of individual AGO2-miRNAs, LIMD1 is most critical at poorly conserved sites, indicating that LIMD1 broadens miRNA regulation beyond ancient, deeply conserved targets. In culture, LIMD1 deficiency de-represses oncogenic proteins that, in vivo, inversely correlate with LIMD1 levels in normal lung and adenocarcinoma, where LIMD1 is characteristically reduced, and whose dysregulation predicts poor survival. Thus, LIMD1 emerges as a key determinant of miRISC architecture, targeting, and potency, challenging RNA-centric models of miRNA function and exemplifying how adaptor proteins diversify post-transcriptional regulation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=180 HEIGHT=200 SRC="FIGDIR/small/680889v2_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@e0f4f5org.highwire.dtl.DTLVardef@f23fd9org.highwire.dtl.DTLVardef@3de3f2org.highwire.dtl.DTLVardef@1404af2_HPS_FORMAT_FIGEXP M_FIG LIMD1 defines the scope of miRNA-mediated targeting and repression O_LIAGO2-chimeric eCLIP in CRISPR-edited human small airway epithelial cells (hSAECs) shows LIMD1 is required for productive AGO2-miRNA engagement transcriptome-wide. C_LIO_LILIMD1 deficiency reduces the AGO2-miRNA:targetome. Each AGO2-miRNA binds fewer targets, with lower occupancy per site and per transcript and fewer global silencing events. C_LIO_LILIMD1 dependence is strongest for GC-poor seed-sites, less conserved miRNAs and sites, and thermodynamically stronger duplexes. C_LIO_LIDosage-dependent effects of LIMD1 deficiency are broadly observed. C_LIO_LITarget-mRNA decay and translational repression are reduced in LIMD1-deficient hSAECs, increasing protein output. C_LIO_LIIn vivo, LIMD1 LOH-associated deficiency is prevalent and typically clonal in NSCLC. LIMD1 expression inversely correlates with oncogene levels in normal lung and adenocarcinoma, and target dysregulation predicts poor survival. C_LI C_FIG

molecular biology↗

bioRxiv2023

A C-terminal HaloTag Impairs AGO2 Function

A 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). Approaches 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 AGO2 biology. Recently, an N-terminal HaloTag-Ago2 fusion 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, permits the creation of endogenous C-terminal tag fusions. 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 AGO2HALO fusion protein has a reduced capacity to interac with the key protein binding partner TNRC6A and that the C-terminal HaloTag does not affect cell viability. However, the AGO2HALO fusion significantly impairs RNA cleavage and RNA silencing activity compared to control cells and reduces nuclear localisation of the fusion protein. Using plasmid constructs and transient transfection, we compared AGO2 tagged with EGFP at the N- or C-terminus in siRNA and miRNA reporter gene assays, and cellular localisation. N-terminally tagged AGO2 functioned and localised similarly to WT untagged AGO2, whereas C-terminally tagged AGO2 was impaired in siRNA and miRNA silencing and exhibited poor nuclear and P-body localisation. We conclude that the fusion of a C-terminal HaloTag to AGO2 is not appropriate for studying AGO2 and RISC. Our results assert the importance of comprehensively validating recombinant tagging strategies to ensure that any experimental results generated do not arise from, or are not obscured by, critical functional defects.

molecular biology↗