RNA’s first letter may shape antiviral alarms, with A outpacing G

Researchers at the International Institute of Molecular and Cell Biology in Warsaw (IIMCB), led by Prof. Gracjan Michlewski, have shown that a subtle difference at the very beginning of an RNA molecule can influence how strongly a cell activates innate immune antiviral responses.

The study, published in Molecular Cell, suggests that RNAs starting with adenosine (A) can trigger a stronger immune response than very similar RNAs starting with guanosine (G). The finding adds an important piece to understanding why some RNAs raise a stronger cellular alarm than others.

How cells recognize dangerous RNA

The study focuses on RIG-I protein, one of the sensors of innate immunity—the body’s first, rapid line of defense against infection. RIG-I detects suspicious RNA molecules, including RNAs linked to viral infection, and helps activate type I interferons, alarm signals that warn the cell and its surroundings of danger.

“We are interested in how cells recognize harmless self RNA from RNA linked to infection or stress. That question is central to understanding antiviral defense, but also to avoiding harmful inflammation when the same defense system is activated at the wrong time,” says Prof. Gracjan Michlewski.

Scientists already knew that RIG-I detects RNAs carrying a 5′-triphosphate group. This group can be understood as a chemical tag that often makes RNA more visible to the immune system. What remained less clear was whether the first nucleotide—the first RNA “letter” directly next to that end—also matters.

The team therefore compared highly similar RNA molecules that differed in this first letter: A or G. The question was not about a large sequence change but about a subtle difference at the very start of the molecule.

A and G: A small difference with a large effect

The results indicated that double-stranded RNAs starting with A activate the RIG-I/type I interferon pathway more strongly than comparable RNAs starting with G. In other words, a single letter can influence how intensely a cell interprets RNA as a potential threat.

Importantly, this difference could not be fully explained by RNA binding to purified RIG-I in a simplified biochemical system. This pointed to a broader explanation: in living cells, immune sensing is shaped not only by the receptor itself but also by the proteins that gather around the RNA.

The team found that RNAs starting with G preferentially recruit GTP-binding proteins. GTP, or guanosine triphosphate, is one of the basic molecules cells use as an energy carrier and as a switch controlling the activity of many proteins.

In this study, the key observation was that GTP-binding proteins may gather around G-starting RNA and partly reduce its recognition by RIG-I. This is the link between the “single-letter” difference and the immune response: the first nucleotide affects not only the RNA molecule itself but also the set of proteins that join it inside the cell.

When G is present at the beginning, the RNA may become partly shielded from the cellular alarm system.

“For us, this is a rewarding result because it shows that a small change at the start of an RNA molecule can strongly affect how cells sense danger. More broadly, the study adds a missing piece to how the early antiviral alarm system works and why some RNAs trigger a stronger response than others, why cellular RNAs are not recognized by this system, and why some viruses can remain undetected by our immune system for a long time,” says Prof. Michlewski.

How the team tested the mechanism

To understand the mechanism, the researchers combined several experimental approaches: synthetic RNAs, cell-based immune assays, RNA sequencing, RNA pull-down coupled with mass spectrometry, biochemical protein–RNA binding tests, microscopy, and measurements of intracellular nucleotides.

RNA pull-down is a method for capturing a chosen RNA molecule together with the proteins bound to it. Coupling this approach with mass spectrometry makes it possible to identify which proteins actually gather around a given RNA in the cell.

Quality control of RNA was also a crucial part of the work. The project changed direction after the team noticed that some earlier results could have been influenced by unwanted by-products generated during RNA synthesis.

In practice, this meant separating the true effects of RNA biology and chemistry from technical effects—signals caused by how the material was produced or purified.

For biotechnology, this is an important process-level message: RNA purity and quality control can determine both how experimental data are interpreted and how final RNA molecules behave in research or therapeutic applications.

The authors stress that the study does not describe a ready-made therapy or diagnostic tool. It investigates a fundamental biological mechanism.

Such mechanisms, however, often become a starting point for later applications—especially in RNA technologies, where it is important to predict whether a molecule should stimulate an immune response or avoid it.

In RNA vaccines and therapeutics, immune activation can be desirable when it strengthens the intended effect or undesirable when it causes excessive inflammation. A better understanding of how the first nucleotide affects immune sensing could help researchers design RNAs with more predictable properties.

“Our results fit this trend by showing a simple rule that may help explain RNA immunogenicity and support safer, more precise therapeutic design. The finding may also have an evolutionary dimension. Many viral RNAs and cellular RNAs differ in the nucleotide found at their 5′ end. Our findings show that this subtle one-letter difference at the start of RNA may have influenced how viral and human RNA evolved over millions of years,” says Prof. Michlewski.

Who’s behind this story?

Sadie Harley

BSc Life Sciences & Ecology. Microbiology lab background with pharmaceutical news experience in oil, gas, and renewable industries.

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Robert Egan

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