mRNA therapeutics—best known through the COVID-19 vaccines—have garnered global attention as a breakthrough technology. Yet, until recently, the precise mechanisms by which these synthetic RNAs operate and are regulated inside cells remained unclear. Driven by the question “What happens when an mRNA vaccine enters the body?”, researchers at the Center for RNA Research within the Institute for Basic Science (IBS) have become the first in the world to uncover the cellular regulatory mechanisms of mRNA therapeutics. In this article, the researchers themselves explain the significance of their findings and key concepts you need to know to understand the study.

mRNA Vaccines and Cellular Regulatory Mechanisms

For decades, the synthesis and intracellular behavior of mRNA have been intensely studied. However, what happens molecularly when synthetic RNA (like that in mRNA vaccines) is introduced into cells from the outside has rarely been investigated. For an mRNA vaccine to work, it must enter the cell safely, be translated into protein, and evade cellular defense mechanisms.

To better understand this process, the IBS team employed CRISPR knockout screening across ~20,000 human genes. They combined this approach with high-purity synthetic mRNA and lipid nanoparticle (LNP) delivery technology, ultimately identifying key molecular players that control the delivery efficiency and stability of mRNA vaccines.

[Figure 1] CRISPR Knockout Screening to Study mRNA Vaccine Regulation
① A knockout library targeting 19,114 human genes was used.
② Synthetic GFP (green fluorescent protein) mRNA—modified with either normal bases or N1-methylpseudouridine—was delivered into knockout cells using lipid nanoparticles.
③ Cells were then sorted by fluorescence intensity, and sequencing was used to identify which genes had been knocked out.
④ The screen revealed critical factors involved in regulating mRNA delivery and expression.

Lipid Nanoparticles and mRNA Delivery

Lipid nanoparticles (LNPs) protect mRNA and deliver it efficiently into cells. The researchers found that heparan sulfate, located on the cell surface, binds to LNPs and facilitates their entry. Once inside, the LNPs are taken into endosomes. There, a proton pump called V-ATPase acidifies the endosome, causing it to rupture. This rupture releases the mRNA into the cytoplasm, where it can finally be translated into protein.

TRIM25 Protein and mRNA Vaccine Defense

However, the cytoplasmic mRNA doesn’t go unnoticed. Cells treat foreign RNA as a threat. The TRIM25 protein quickly recognizes and binds to the mRNA, working with other enzymes and cofactors to degrade it. This is where N1-methylpseudouridine, a base modification used in COVID-19 mRNA vaccines, comes in. This modification helps inhibit TRIM25’s ability to bind and degrade the mRNA, allowing it to evade immune detection and remain stable for protein production. This innovation significantly improved vaccine efficacy and was recognized with the 2023 Nobel Prize in Physiology or Medicine.

The Role of Protons: A New Immune Insight

The study also revealed a new finding: the protons released by V-ATPase activate TRIM25. In an acidic environment, TRIM25 binds more strongly to its target mRNA. This is the first report showing that changes in cytoplasmic pH directly regulate RNA immune responses, offering novel insights into how cells defend against foreign genetic material.

[Figure 2] Key Regulatory Pathways and the Role of Modified Bases
① Delivery Regulation
Heparan sulfate facilitates LNP entry. V-ATPase acidifies the endosome, causing it to rupture and release mRNA into the cytoplasm.
② TRIM25 Suppression of Foreign mRNA
Protons released during rupture activate TRIM25, which binds and degrades the mRNA—a pH-dependent defense mechanism.
③ Effect of N1-methylpseudouridine:
This modification weakens TRIM25’s binding to mRNA, helping it avoid detection and degradation. COVID-19 mRNA vaccines using this technique showed greatly improved efficacy.

Applications and Future Research

The team now plans to identify the RNA sequence and structural features preferred by TRIM25 to guide the development of next-generation mRNA therapeutics that are both safer and more effective. In addition, they aim to expand research on how proton levels regulate cell function, with the goal of further optimizing mRNA drug delivery and modulating immune responses.