Our immune system relies on many different cell types working together to detect and respond to threats. One important group of immune cells is monocytes, which help recognize invading microbes and trigger inflammatory responses. Although scientists have long known that gene expression changes during immune activation, the role of alternative splicing in this process is still being uncovered.

Researchers from University Medical Center Utrecht and Utrecht University in the Netherlands used native long-read RNA sequencing to examine how alternative splicing changes when human monocytes become activated.

Alternative splicing is a process that allows a single gene to produce multiple RNA transcripts, known as isoforms. These different isoforms can generate proteins with distinct structures and functions, greatly increasing the diversity of molecules that cells can produce.

To investigate this process, the researchers used Oxford Nanopore native RNA sequencing to analyze primary human monocytes before and after stimulation with lipopolysaccharide, or LPS, a bacterial molecule commonly used to activate immune cells.

The long-read sequencing approach allowed the team to capture complete RNA molecules rather than short fragments. This provided a much more detailed view of transcript structure and enabled the identification of individual isoforms.

The researchers identified more than 24,000 expressed isoforms, including thousands that had not previously been annotated. Following immune activation, monocytes showed widespread changes in isoform expression and extensive isoform switching events.

Importantly, these changes were not random. Activated monocytes preferentially expressed longer isoforms that were more likely to produce functional proteins. These transcripts contained complete open reading frames, fewer retained introns, and greater protein domain complexity.

To verify their findings, the researchers compared the long-read results with conventional short-read RNA sequencing data and found strong agreement between the two approaches.

The team also integrated ribosome profiling and proteomics data to determine whether the altered RNA isoforms were being translated into proteins. The results showed that the activation-associated isoforms were linked to increased production of immune effector proteins, suggesting that alternative splicing directly contributes to immune function.

These findings indicate that alternative splicing is an active regulatory mechanism during immune activation rather than simply a byproduct of gene expression changes. By selecting specific isoforms, monocytes appear to fine-tune the proteins they produce in response to infection or inflammation.

The study also highlights the value of long-read RNA sequencing for understanding transcript diversity. Traditional short-read approaches often struggle to reconstruct complete transcript isoforms, while long-read technologies provide a clearer view of how RNA molecules are organized.

As long-read RNA sequencing becomes more widely adopted, researchers may uncover many additional isoform-level regulatory mechanisms that influence immunity, disease progression, and therapeutic responses. Understanding these transcript-level changes could ultimately lead to improved biomarkers and new strategies for modulating immune function.