The empty octopus in the cell Researchers identify how the protein Argonaute is targeted for recycling to maintain microRNA function

Autophagic degradation of empty Argonaute by VCP. Argonaute proteins, which were named after the Argonauta octopus, are stable when loaded with microRNAs but are selectively degraded when they are empty. Researchers revealed that empty Argonaute (blue balloon) is degraded by macroautophagy (recycling bin) via VCP machinery (little girl), which ensures proper microRNA function. ©︎ 2019 Aoha, Wakana and Hotaka Kobayashi.

A fundamental research study on microRNAs, tiny molecules that help control gene activity inside plants and animals, has made an unexpected connection to a molecule associated with multiple genetic diseases.

Researchers at the Institute for Quantitative Biosciences discovered that a protein, VCP, known for its involvement in neurodegenerative diseases also maintains the cell's healthy microRNA machinery.

That microRNA machinery is the protein Argonaute, named after the Argonauta octopus, also known as paper nautiluses. Argonaute proteins and microRNAs form a functional complex and control how genes are expressed.

The same research team previously identified and named the protein Iruka (the Japanese word for dolphin), which adds a "tag" to microRNA-free — or empty — Argonaute so it can be eliminated. That tag is a tiny protein called ubiquitin.

"Our previous study revealed that removal of empty Argonaute is a form of quality control to eliminate dysfunctional Argonaute. This action ensures efficient microRNA function," said Professor Yukihide Tomari, last author of the recent research publication.

However, Iruka adding a ubiquitin tag to empty Argonaute is just the first step in a chain of events that leads to the eventual breakdown and recycling of Argonaute proteins. Researchers continued following the trail of empty Argonaute through the cell to understand the full degradation pathway.

The research team first collected empty Argonaute proteins from fruit fly cells. The fruit fly is an organism well-suited for studying the Argonaute pathway because it only has one type of Argonaute protein that loads microRNAs, while mammals have four.

Then, researchers used a technique that can identify protein fragments based on their size and weight, called liquid chromatography tandem mass spectrometry (LC-MS/MS). One of the common proteins bound to ubiquitin-tagged Argonaute was VCP, valosin-containing protein.

In its known roles, VCP identifies proteins tagged with ubiquitin and sends them to be degraded. Other research teams have discovered that different mutations in VCP can cause a variety of genetic disorders including Parkinson's disease, amyotrophic lateral sclerosis, also known as ALS or Lou Gehrig’s disease, and inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), a condition which affects the muscles, bones and brain.

Researchers now have a more detailed understanding of the Argonaute portion of microRNA regulation. Iruka identifies Argonaute proteins that are not carrying microRNAs. Iruka adds ubiquitin to those empty Argonaute proteins, which attracts the attention of VCP. VCP sends the empty Argonaute to be broken down via macroautophagy, one of the cell's recycling mechanisms.

A cartoon version of the original photo above. ©︎ 2019 Caitlin Devor, University of Tokyo, CC-BY.

Connecting VCP to microRNA gene regulation via the Argonaute pathway may help explain why mutations in VCP can cause such diverse human diseases.

"We conducted this study purely based on our scientific curiosity," said Hotaka Kobayashi, Ph.D., first author of the recent research publication. Kobayashi was a research associate at the time of the research and is currently a research fellow at the Albert Einstein College of Medicine in the U.S.

"Although additional studies are needed to clarify the potential connection between VCP-related disorders and microRNAs, this research epitomizes the possible connections between basic and applied research," he said.

Researchers plan to continue mapping the details of the Argonaute recycling pathway to understand its role in microRNA regulation.

Collaborators from Utsunomiya University also contributed to this research.

Funding was provided by the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT), the Japan Society for the Promotion of Science (JSPS) and the Naito Foundation, Japan.