RNA therapeutics have emerged as one of the most promising new classes of medicines. Eight small interfering RNA (siRNA) drugs have already been approved worldwide for the treatment of genetic diseases, yet scientists have not fully understood one of the most fundamental steps underlying their function: how Argonaute, the core protein responsible for gene silencing, becomes activated.
A research team led by Director KIM V. Narry at the Center for RNA Research within the Institute for Basic Science (IBS), together with Professor ROH Soung-Hun at Seoul National University, has now revealed for the first time how Argonaute acquires its functional form by loading small RNAs.
MicroRNAs (miRNAs) are small RNAs that help regulate gene expression, while RNA therapeutics using siRNAs exploit the same biological mechanism to silence disease-causing genes. Argonaute serves as the central component of the RNA-induced silencing complex (RISC), a molecular machine that uses these small RNAs to identify and silence specific genes. Much like a detective uses clues to identify a suspect, Argonaute relies on small RNAs to locate specific target genes among thousands of possibilities within the cell.
However, despite its central role in gene silencing, researchers have not fully understood how bulky miRNA are loaded into Argonaute and assembled into an active gene-silencing complex. This knowledge gap has limited efforts to design more effective RNA therapeutics.
One longstanding mystery is how Argonaute adopts a tightly packed structure that appears too narrow to accommodate the bulky miRNA that must be loaded during RISC assembly. Scientists knew that molecular chaperones assist this process, but the underlying mechanism had remained elusive.
To address this question, the researchers successfully isolated and purified a previously unknown Argonaute maturation complex (AMC), in which Argonaute is associated with the chaperone proteins Hsp90. Using state-of-the-art cryo-electron microscopy (cryo-EM), they determined the three-dimensional structure of the complex at near-atomic resolution.
The structure revealed that chaperone proteins hold Argonaute in an unusually open conformation, creating enough space for a miRNA to enter. Once the miRNA is loaded, the chaperones are released and Argonaute folds into its mature functional form capable of silencing genes.
Unexpectedly, the study showed that the RNA itself plays a far more active role than previously recognized. The researchers found that miRNA is not merely cargo delivered to Argonaute. Instead, the miRNA acts as a molecular cofactor that helps guide Argonaute folding. In the absence of a proper miRNA, Argonaute failed to acquire its functional structure. This finding suggests that RNA itself participates directly in the protein folding process.
The team further identified key structural features required for efficient Argonaute assembly. They showed that miRNA must possess specific chemical characteristics, retain a two-stranded structure, and have an optimal length of approximately 20–24 nucleotides. These properties closely match those found in naturally occurring miRNAs and therapeutic siRNAs.
Importantly, the researchers established an experimental system that faithfully reproduces Argonaute loading and RISC assembly outside living cells. Using this platform, they directly examined how chemical modifications commonly used in therapeutic siRNAs affect Argonaute assembly and function.
"This achievement provides, for the first time, a molecular basis for RNA therapeutic design, which has until now relied largely on trial and error," said Director KIM V. Narry, co-corresponding author of the study. "We expect these findings will contribute to the development of safer and more effective RNA therapeutics."
Professor ROH Soung-Hun, co-corresponding author of the study, added, "Researchers have traditionally focused on protein structures after they have already formed. In this study, we were able to directly observe the process by which a protein acquires its function. The findings provide a new perspective on how chaperones and RNA cooperate to create functional biological molecules."
Beyond advancing understanding of RNA biology, the study establishes a new framework for rational RNA therapeutic design and provides broader insight into how molecular chaperones cooperate with biological ligands to guide protein folding.
The findings were published in Nature on June 10.
