FOCUS Biochemistry It's an RNA World Again The Surprising Role of RNA on a Cell's Surface By Kenny Cheng and Risha Chakraborty Art by Luna Aguilar 22 Yale Scientific Magazine March 2024 www.yalescientific.org
Ribonucleic acids (RNA) are among the most important molecules in the human body. Early in the evolution of life, RNA controlled all molecular and cellular functions, including the catalysis of biochemical reactions. Today, RNA carries out myriad functions within cells, such as communicating genetic information from DNA to functional protein products, regulating gene expression, and defending cells against viral infections. Recently, however, it has been discovered that RNA is not only confined within the cell but also present on the cell’s surface. RNA…Outside of Cells? In 2011, RNA was first discovered binding to the cell membrane—which separates a cell’s interior from the outside—in bacteria in the lab of Ronald Breaker, a Sterling Professor of Molecular, Cellular, and Developmental Biology at Yale. It was not until 2020 that Liangfang Zhang and Sheng Zhong, professors of bioengineering at the University of California, San Diego, discovered what they coined “membrane-associated extracellular RNAs.” These “maxRNAs” were RNAs stably associated with the outer membrane surface of human cells, presenting a mysterious new role of RNAs. This discovery was quickly compounded upon by Nobel Laureate Carolyn Bertozzi in a 2021 paper announcing the discovery of “glycoRNAs,” small extracellular noncoding RNAs with special sugar molecules attached to them. These recent discoveries opened the door for further exploration into the functions of extracellular RNAs. When Jun Lu, an associate professor of genetics at the Yale School of Medicine (<strong>YSM</strong>), learned of these findings, he was surprised by the reported stability of the extracellular RNAs. “Outside of cells, we know that there are lots of RNases that act as Pac-Mans to chew up extracellular RNAs very efficiently,” Lu said. Indeed, the idea that RNAs could exist outside of cells for prolonged periods posed a confounding mystery. Interested in uncovering the functional roles of glycoRNAs, Lu approached his upstairs lab neighbor, frequent collaborator, and expert on neutrophil biology, Dianqing (Dan) Wu, the Gladys Phillips Crofoot Professor of Pharmacology at <strong>YSM</strong>. The result was a study affirming the presence of extracellular RNAs on neutrophils and their function in facilitating neutrophil recruitment to infection sites. Wenwen Tang (front row, left), Ningning Zhang (front row, right), Jun Lu (back row, left), and Dan Wu (back row, right) pose for a picture in their laboratory. The Firefighters of the Blood Stream Neutrophils are the most common white blood cell, or infection-fighting cell, in the human bloodstream. While extensive research has been done on their immune function at infection sites, less is known about how they navigate to these locations. Traditionally, neutrophil biologists have focused on the process of rolling adhesion as an important step for this movement. Neutrophils typically float in the bloodstream untethered to any scaffolding. But when injury or inflammation exposes the cell adhesion molecules on endothelial tissue lining blood vessels, neutrophils adhere to the walls of blood vessels and roll toward the site of injury to fight accumulating pathogens. Before this collaboration, Wu didn’t have much experience in RNA research. Being a neutrophil biologist, however, he did have the intuition that something in the neutrophil-endothelial interaction included sugar-binding. Neutrophil biologists had previously identified a class of proteins found on endothelial cells called selectins that appeared to bind proteins with attached sugars on the surface of neutrophils. These interactions were reversible, allowing neutrophils to swiftly engage with endothelial tissue repeatedly, which underlies the ability of these cells to roll along blood vessel walls. To investigate the presence of cell surface RNAs in this process, the authors utilized a sophisticated technique called click chemistry, which won Bertozzi her Nobel Prize in Chemistry in 2022. Click chemistry describes reactions that are simple, highly efficient, and selective, just like clicking a button. Ideally, these reactions should also avoid side reactions and provide high yields of the PHOTOGRAPHY BY EMILY POAG desired product at mild conditions. Click chemistry is often used to join molecular building blocks together with applications in bioconjugation, materials science, and drug discovery. In this study specifically, the authors employed a bioorthogonal reaction, which is a click reaction in a biochemical setting that selectively targets biological molecules without disturbing other cellular processes. First, the authors labeled the RNA of cells with a sialic acid sugar mimetic called Ac4ManNAz, which acts as a molecular tag, forming glycoRNAs. Then, using click chemistry, they attached a biotin molecule to the labeled sugars in the glycoRNAs. The attached biotin subsequently served as a beacon, allowing glycoRNAs to be easily detectable. The researchers found a strong biotin signal on the surface of neutrophils, suggesting the startling presence of glycoRNA. They then confirmed that glycoRNAs bind to a specific selectin called P-selectin on endothelial cells. By binding P-selectin, glycoRNAs help facilitate the neutrophil-endothelial interaction necessary for neutrophils to roll along blood vessels. The combination of discovering the glycosylated ligands on the neutrophil cell surface and elucidating at least one functional role of extracellular RNA involved a fair bit of serendipity. “I think both of us were a little skeptical initially [at the magnitude of the effects of removal of extracellular RNAs]. We wanted to make sure this wasn’t just a single-time interaction,” Wu said. Together, Wu and Lu’s labs were able to demonstrate the functional importance of neutrophil cell surface RNAs dozens of times, giving them the green light in terms of reproducibility. www.yalescientific.org March 2024 Yale Scientific Magazine 23