Pseudouridine (Ψ) is one of the most common RNA modifications in human cells, introduced post-transcriptionally by enzymes known as pseudouridine synthases (PUS). These modifications act as punctuation in the cell’s genetic text - small chemical notations that help ensure RNA messages are read correctly and translated into functional proteins, with key roles in translation, splicing, and RNA stability.
Although essential for the proper functioning of cells, the enzymes that install these notations - and their precise roles in health and disease - have remained poorly understood due to limited knowledge linking specific PUS enzymes to their RNA targets.
To address this gap, researchers at Ludwig Oxford and the Target Discovery Institute, led by Chunxiao Song and Parinaz Mehdipour, have created the first comprehensive map of PUS-dependent modifications in human RNA, providing new insight into how these molecular changes might influence gene expression and disease.
To achieve this, the team systematically generated knockouts and knockdowns of individual PUS enzymes and then mapped the resulting pseudouridine profiles using a powerful sequencing method developed in the Song lab, known as BACS (2-bromoacrylamide-assisted cyclisation sequencing). BACS enables direct, quantitative, base-resolution sequencing of pseudouridine by introducing Ψ-to-C mutations - enabling the researchers to pinpoint where and when each enzyme adds pseudouridine across different types of RNA.
Their results have produced a map of pseudouridine sites in human transfer RNAs (tRNAs), uncovered targets dependent on PUS enzymes, and showed that individual PUS enzymes act at distinct stages of RNA processing.
Pseudouridine synthases are increasingly recognised as key players in genetic diseases and cancer, yet their specific biological functions have been difficult to define. By linking each enzyme to its precise RNA targets, this new study provides a foundational reference for understanding how these enzymes contribute to human biology - and how their disruption may drive disease.
Chunxiao said:
“I’m delighted that we used our BACS method to address an important question in pseudouridine biology. Together with Pari’s group, we identified the target pseudouridine sites for all 13 human pseudouridine synthases. This map offers a valuable resource for understanding how pseudouridine and these enzymes function in human biology, including their roles in cancer.”