Study reveals “two-factor authentication” system that controls microRNA destruction

Cells rely on tiny molecules called microRNAs to tune which genes are active and when. Cells must carefully control the lifespan of microRNAs to prevent widespread disruption to gene regulation.A new study led by researchers at MIT’s Whitehead Institute for Biomedical Research and Germany’s Max Planck Institute of Biochemistry reveals how cells selectively eliminate certain microRNAs through an unexpectedly intricate molecular recognition system. The open-access work, published on March 18 in Nature, shows that the process requires two separate RNA signals, similar to how many digital systems require two forms of identity verification before granting access.The findings explain how cells use this “two-factor authentication” system to ensure that only intended microRNAs are destroyed, leaving the rest of the gene regulation machinery in operation.MicroRNAs are short strands of RNA that help control gene expression. Working together with a protein called Argonaute, they bind to specific messenger RNAs — the molecules that carry genetic instructions from DNA to the cell’s protein-making machinery — and trigger their destruction. In this way, microRNAs can reduce the production of specific proteins.While scientists recognized that microRNAs could be destroyed through a pathway known as target-directed microRNA degradation, or TDMD, the details of how cells recognized which microRNAs to eliminate remained unclear.“We knew there was a pathway that could target microRNAs for degradation, but the biochemical mechanism behind it wasn’t understood,” says MIT Professor David Bartel, a Whitehead Institute member and co-senior author of the study.Earlier work from Bartel’s lab and others had identified a key player in this pathway: the ZSWIM8 E3 ubiquitin ligase. E3 ubiquitin ligases are involved in the cell’s recycling system and attach a small molecular tag called ubiquitin to other proteins, marking them for destruction.The researchers first showed that the ZSWIM8 E3 ligase specifically binds and tags Argonaute, the protein that holds microRNAs and helps regulate genes. The researchers’ next challenge was to understand how this machinery recognized only Argonaute complexes carrying specific microRNAs that should be degraded.The answer turned out to be surprisingly sophisticated.Using a combination of biochemistry and cryo-electron microscopy — an imaging technique that reveals molecular structures at near-atomic resolution — the researchers discovered that the degradation system relies on a dual-RNA recognition process. First, Argonaute must carry a specific microRNA. Second, another RNA molecule called a “trigger RNA” must bind to that microRNA in a particular way.The degradation machinery activates only when both signals are present.This dual requirement ensures exquisite specificity. Each cell contains over a hundred thousand Argonaute–microRNA complexes regulating many genes, and destroying them indiscriminately would disrupt essential biological processes.“The vast majority of Argonaute molecules in the cell are doing useful work regulating gene expression,” says Bartel, who is a professor of biology at MIT and also a Howard Hughes Medical Institute investigator. “You only want to degrade the ones carrying a particular microRNA and bound to the right trigger RNA. Without that specificity, the cell would lose its microRNAs and the essential regulation that they provide.”The structural images revealed complex molecular interactions. The ZSWIM8 ligase detects multiple structural changes that occur when the two RNAs bind together within the Argonaute protein.“When we saw the structure, everything clicked,” says Elena Slobodyanyuk, a graduate student in Bartel’s lab and co-first author of the study. “You could see how the pairing of the trigger RNA with the microRNA reshapes the Argonaute complex in a way that the ligase can recognize.”Beyond explaining how TDMD works, the findings may impact how scientists think about the regulation of RNA molecules more broadly.“A lot of E3 ligases recognize their targets through simpler signals,” says Jakob Farnung, co-first author and researcher in the Department of Molecular Machines and Signaling at the Max Planck Institute of Biochemistry. “It was like opening a treasure chest where every detail revealed something new and mesmerizing.”MicroRNAs typically persist in cells for much longer time periods than most messenger RNAs, but some degrade far more quickly, and the TDMD pathway appears to account for many of these unusually short-lived microRNAs.The researchers are now investigating whether other RNAs can trigger similar degradation pathways and whether additional microRNAs are regulated through variations of the mechanism shown in this study.“This opens up a whole new way of thinking about how RNA molecules can control protein degradation,” says Brenda Schulman, study co-senior author and director of the Department of Molecular Machines and Signaling at the Max Planck Institute of Biochemistry. “Here, the recognition was far more elaborate than expected. There’s likely much more left to discover.”Uncovering the details of this intricate regulatory system required interdisciplinary collaboration, combining expertise in RNA biochemistry, structural biology, and ubiquitin enzymology to solve this long-standing molecular puzzle.“This was a project that required the strengths of two labs working at the forefront of their fields,” says Schulman, who is also an alum of Whitehead Institute. “It was an incredible team effort.”