New publication

Study title: Neurodevelopmental disorder–linked Argonaute mutations permit delayed RISC assembly and impaired microRNA-mediated gene regulation.

This study investigates possible underlying molecular mechanism of AGO syndromes.

Quick explainers

Cryo-electron miscroscopy (cryo-EM):
Imaging method that freezes proteins at extremely low temperatures and then photographs them with an electron beam. This allows to see 3D structures of proteins.

RISC (RNA-Induced Silencing Complex):
A molecular “gene-regulation machine” including Argonaute + a microRNA guide strand.
RISC turns specific genes on or off to help control e.g. brain development.

Passenger strand:
Argonaute must remove the passenger strand from an incoming microRNA duplex so the guide strand can regulate genes. Of the two strands in the duplex,

• one strand becomes the guide (the useful one that will regulate genes),

• the other is the passenger (the helper strand that must be removed).

Exonuclease:
An enzyme that trims RNA or DNA from either of the ends. Exonucleases ISG20, TREX1, and ERI1 regulate microRNA (miRNA) stability and activity by trimming miRNA when bound to Argonaute proteins in RISC.

What did the researchers find?

1. The mutant protein looks structurally normal

Using high-resolution cryo-electron miscropy (cryo-EM), researchers found that AGO1(ΔF180) keeps the overall 3D architecture of the normal Argonaute protein.
However, the mutation removes a stabilizing interaction in the protein’s core. Nearby amino acids unexpectedly shift to compensate, causing the protein to look normal and recognise guide RNAs.

Fig. 1A-D: AGO1(ΔF180) retains the ability to form functionally mature RISCs. Savidge et al. PNAS 2025

2. RISC function fails due to a “duplex-bound” stall

The researchers next asked: Does the mutation affect how Argonaute assembles a working RISC complex?

Mutant AGO1 proteins (ΔF180 and L190P):

• fail to efficiently eject the passenger strand, which

• exposes miRNA to exonucleases that trim the RNA ends,

• and produces truncated guide.

This prevents the protein from maturing into a functional RISC complex and Argonaute cannot regulate its target genes.

The paper’s Fig. 2G illustrates this: the mutant remains stuck in a prolonged duplex-bound intermediate.

Fig. 2G: Schematic of RISC assembly model of AGO syndrome mutants leading to shortened guide RNAs. Savidge et al., PNAS 2025

3. Trimming defects occur across multiple AGO mutations

The same problem was seen in additional patient-associated variants, including AGO1(G199S), AGO2(ΔF182), and AGO2(G201V).

This indicates a shared disease mechanism: Argonaute mutants stay bound to microRNA duplexes too long, leading to aberrant trimming and failure of functional RISC assembly.

4. Under stress or viral infection it may get worse

Under cellular stress or viral infection, the exonuclease ISG20 is strongly induced, which could further increase trimming and lead to even fewer working RISC complexes.

In summary

• The AGO1(ΔF180) mutation keeps the protein’s overall shape, but breaks its function.

• The mutant protein fails at efficient passenger strand ejection. It holds on to duplex microRNAs too long and blocks the formation of normal RISC complexes.

• This leads to aberrant trimming of microRNAs and failure of gene regulation.

• Several studied AGO1 and AGO2 mutations share this same problem.

• During stress or viral infection, this failure could become even worse.

• Mutant proteins may act as “miRNA sponges” or “dead‐end” complexes, which trap microRNAs instead of letting them regulate genes.

• This leads to fewer usable microRNAs in the cell, loss of proper gene regulation, and widespread downstream effects on brain development.

References

Savidge A, Zhang H, Annasaheb Adhav V, Kehling AC, Sim G, Shen Z, Fu TM, Nakanishi K., Neurodevelopmental disorder–linked Argonaute mutations permit delayed RISC formation and unusual shortening of miRNAs by 3′→5′ trimming. Proc Natl Acad Sci U S A. 2025 Nov 18;122(46):e2524644122.