WBP2/YAP1 complex governs miR-133b maturation and ovarian development in teleosts

Fish are not just simple creatures of the deep; they are complex biological systems where the development of their reproductive organs relies on a delicate, invisible conversation between different types of molecules. In the world of vertebrate biology, a specific type of molecule called a microRNA acts as a crucial regulator. Think of these as tiny managers that do not build the cell's machinery themselves but instead decide which instructions get read and which are silenced. In fish, these managers are essential for the ovaries to mature correctly, ensuring that eggs develop at the right time. One specific manager, known as miR-133b, has been known to target and suppress a protein called WBP2, which is involved in making estrogen, the hormone that drives female development. For a long time, scientists believed this relationship was a one-way street: the manager silences the protein. However, biology is rarely so simple, and the question remained whether the protein could talk back to the manager to control its own production.

A team of researchers from the Southern Marine Science and Engineering Guangdong Laboratory in Zhanjiang set out to solve this puzzle using two species of fish: the greater amberjack and the large yellow croaker. They wanted to understand how the ovaries of these fish mature and whether the relationship between the manager and the protein was actually a two-way conversation. Their work reveals that the protein WBP2 does not just sit passively waiting to be silenced. Instead, it teams up with another protein called YAP1 to form a complex that actively helps produce the very manager that controls it. This discovery changes our understanding of how fish reproduce, showing that the system relies on a closed loop where the parts constantly adjust each other to maintain balance.

To uncover how these molecules interact, the scientists first needed to see if the proteins could physically hold onto one another. They produced large quantities of the WBP2, YAP1, and a third protein called DGCR8, which is part of the machine that creates microRNAs. By mixing these proteins in a test tube and using a technique that acts like a molecular fishing rod, they confirmed that WBP2 binds directly to both YAP1 and DGCR8. To see exactly where they connected, the researchers used a method called cross-linking mass spectrometry, which essentially freezes the proteins in place and maps their contact points. They found specific spots on the proteins where they lock together, proving that WBP2 acts as a scaffold, a structural bridge that brings YAP1 and the microRNA-making machine together. Computer models supported this, showing that the three proteins fit together to form a stable unit.

The researchers then tested what happens when this team is broken apart. They used a technique to silence the genes for WBP2 and YAP1 in fish cells, effectively turning them off. When they silenced just one of the proteins, the production of the miR-133b manager dropped significantly. But when they silenced both at the same time, the effect was even stronger, suggesting that the two proteins work together as a team to ensure the manager is made. This confirmed that WBP2 and YAP1 are not just passive targets but are active partners in the creation of the microRNA. The study also showed that this process is specific; the proteins do not just randomly affect all microRNAs, but they specifically help mature miR-133b.

The story does not end in the test tube. The team wanted to see how this molecular teamwork affects the actual fish. They injected large yellow croaker with a substance to silence the WBP2 gene and then examined the ovaries under a microscope. In normal fish, the ovaries contain many small, developing eggs. In the fish where WBP2 was silenced, the ovaries looked different; they contained larger, more mature eggs with more yolk, indicating that the ovaries had rushed ahead in their development. This suggests that when the WBP2-YAP1 team is missing, the balance is lost, and the ovaries mature too quickly. This observation supports the idea that the normal job of this protein team is to carefully regulate the production of the miR-133b manager, which in turn keeps the estrogen levels in check.

To understand the full picture, the researchers also looked at how the fish responded to estrogen itself. They treated the fish with extra estrogen and with a drug that blocks estrogen, then measured the levels of the manager and the proteins in the brain and ovaries. They found that in female ovaries, adding estrogen caused the fish to produce more of the miR-133b manager, which then went on to suppress the genes that make estrogen. This creates a self-correcting loop: when there is too much estrogen, the fish makes more managers to turn it down. The blocking drug produced the opposite effect, changing the levels of the manager and the proteins in a way that confirmed this system is sensitive to the body's hormonal state. The results showed that this regulatory circuit is not just a simple line but a complex network that involves the brain and the reproductive organs, and it behaves differently in males and females.

The findings from this study provide a clear view of a previously hidden mechanism in fish biology. It shows that the relationship between miR-133b and WBP2 is not a one-way command but a reciprocal feedback loop. The protein WBP2, working with YAP1, helps create the manager miR-133b, and that manager then turns down the production of the protein. This cycle ensures that the ovaries develop at the right pace and that estrogen levels remain stable. By mapping the physical connections between these proteins and observing how the fish change when the system is disrupted, the researchers have filled a gap in our understanding of how vertebrates control their reproduction. This work suggests that the ability to fine-tune gene expression through such loops is a fundamental part of how life maintains its balance, offering new insights into the complex machinery that drives the development of fish and potentially other animals.