Unsealing cells’ ‘black box’ strategy to regulate gene activation

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Unsealing cells' 'black box' strategy to regulate gene activation

Researchers identify step-by-step RNA interference process

While scientists have known for over two decades that all cells use a strategy called RNA interference to regulate gene expression, a new study is the first to describe how a specific protein manages the step-by-step process of assembling the molecular complex that performs the regulatory job.

Among the surprising findings: The messenger RNA (mRNA) that is targeted for interference - suppression of the protein whose instructions are carried by this same mRNA - also helps with completion of the final step of assembling the complex that inhibits gene expression.

Overall, the study shows that the managing protein undergoes a four-step process to convert a precursor complex into a mature RNA-induced silencing complex, or RISC.

"This mechanism has been a black box for a quarter century, since the discovery of RNA interference," said senior author Kotaro Nakanishi, professor of chemistry and biochemistry at The Ohio State University.

"Visualizing the mechanism is very important - pharmaceutical companies may appreciate our 3D structures because they can optimize or design a new drug based on them."

The study is published today (May 26, 2026) in the journal Molecular Cell.

Nakanishi outlined in a 2022 paper how the "superfamily" of four Argonaute proteins is involved in RNA interference, explaining that microRNAs and small interfering RNAs (siRNAs), both small segments of RNA that inhibit genes' protein-building functions, must be loaded into Argonaute proteins to carry out that job - they cannot perform the task on their own.

In this new work, Nakanishi and colleagues conducted biochemical assays and cryogenic-electron microscopy using Argonaute2 as a model, "but based on the biochemical data, we believe that all of the Argonaute proteins behave the same way," he said.

The starting point of the study involved incubating a human Argonaute2 protein with a specific siRNA duplex.

The results identified a series of steps that followed: Argonaute2 loads the double-stranded RNA and selects one strand over another to serve as a guide. The protein then unwinds the duplex and ejects the "passenger" strand that's no longer of use.

The ejection revealed the surprising role for the target mRNA - helping cast off the passenger strand - which led the team to call the final step TAPE, for target-assisted passenger ejection.

Though mRNAs have been viewed primarily as targets of microRNA and siRNA in most instances of RNA interference, "our results instead suggest that mRNAs can also bind precursor RISCs and facilitate passenger removal during RISC maturation," the authors wrote.

"It's complicated, but it also makes sense to me that if the cell is targeting predominant mRNAs, these targeted mRNAs also facilitate formation of the RISC," Nakanishi said.

He said the findings should help advance development of therapeutic siRNAs and cityRNAs (cleavage-inducing tiny RNAs) that could be designed to override natural cellular processes and prompt a specific RNA interference process to silence problematic genes linked to diseases.

"Researchers and pharmaceutical companies have been using siRNAs as a potential therapy to shut down gene expression and study the role of the protein of interest. However, nobody knows how RISCs are formed," Nakanishi said. "So now we can provide a robust structural basis or foundation to design or optimize siRNAs."

His lab is now using the same techniques to confirm how the other three Argonaute proteins assemble RISCs.

This research was supported by the National Institutes of Health and fellowships from Ohio State's Center for RNA Biology.

Co-authors included Huaqun Zhang, Vishal Annasaheb Adhav, Audrey Kehling, Andrew Savidge and Zhangfei Shen, all of Ohio State, and Tianmin Fu of the University of Massachusetts Chan Medical School. Giovanna Grandinetti and Yohie Narui of Ohio State's Center for Electron Microscopy and Analysis helped with collection of high-quality cryo-EM micrographs.

Nakanishi is a co-founder and scientific advisor of and reports a financial interest in City Therapeutics, Inc.

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