# Dysregulation of AGO2-miRNA dynamics underlies the AGO2-associated Lessel–Kreienkamp syndrome

## TL;DR

This study shows how specific mutations in the AGO2 protein disrupt its normal function, leading to neurodevelopmental disorders by altering miRNA dynamics.

## Contribution

The study reveals that AGO2 mutations cause gain-of-function effects through altered miRNA targeting and processing dynamics.

## Key findings

- Certain AGO2 mutants show prolonged dwell times on target RNAs, impairing target release.
- In neurons, the L192P mutation alters miRNA loading and increases 3′ isomiR accumulation.
- Mice with the L192P mutation exhibit reduced breeding ability and altered cortical transcriptomes.

## Abstract

Mutations in human Argonaute genes, AGO1 and AGO2, are associated with neurodevelopmental disorders. Although multiple patients have been identified, the underlying molecular basis for pathogenesis remains unclear. Here, we biochemically examined five AGO2 mutations (p.L192P, p.A367P, p.T357M, p.F182del, and p.G733R) linked to different clinical severities. Except for G733R, all AGO2 mutant proteins maintained a stable fold, capable of binding and using microRNA (miRNA) guides. Kinetic studies showed that the L192P, A367P, and T357M mutants have prolonged dwell times on target RNAs, indicating impaired target release. The L192P and A367P variants also display slow target RNA association kinetics. RNA Bind-n-Seq experiments showed that in vitro, the L192P, A367P, T357M, and F182Δ mutants are prone to mis-targeting. In cultured murine cortical neurons, the L192P mutant altered the miRNA complement associated with AGO2, altered guide strand selectivity, and increased the accumulation of 3′ isomiRs, suggesting altered miRNA loading and increased miRNA 3′ end exposure. In vivo, mice carrying the p.L192P variant, but not p.G733R, demonstrated strongly reduced breeding ability, altered cortical transcriptomes, and over-repression of miRNA targets. The combined results suggest patient mutations impact AGO2 targeting dynamics in a gain-of-function manner, leading to deregulation of the neuronal transcriptome and the observed neurodevelopmental anomalies.

Graphical Abstract