New study by Nakanishi lab shows that AGO mutants keep the protein's shape, but possibly act as “miRNA sponges"
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. It was inspired by the first Argonautes-AGO Syndromes meeting in 2022, a testament to the catalyst nature of these meetings. We would like to express our gratitude to Prof. Nakanishi for his dedication.
I first learned about AGO syndrome at the inaugural Argonaute meeting in Germany, where I also met several families whose children were affected. Around the same time, my group discovered that specific exonucleases—some of which are induced under stress or during viral infection—can shorten miRNAs from their standard length (~22 nucleotides) to ~14 nucleotides. Since then, we have been investigating a possible connection between AGO syndrome and viral infection.
Kotaro Nakanishi
Professor at Ohio State University
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 one end of miRNAs loaded into Argonaute proteins within RISC.
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.
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.
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.
Under cellular stress or viral infection, the exonuclease ISG20 is induced, which could further increase trimming. This yet needs to be experimentally validated.
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.
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.
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.