Trub1-mediated pseudouridylation is dispensable for immune cell development and homeostasis

Epitranscriptomic modifications are evolutionarily conserved changes to RNA and are present in all RNA types, including mRNA. Next to methylation, pseudouridine is also a prevalent modification found in mRNA and other RNA types. Pseudouridylation of RNA improves RNA stability, RNA stacking and translation fidelity. While m⁶A RNA methylation has been demonstrated to regulate the activation of various immune cell types, such as T cells, dendritic cells or NK cells, insight into the role of pseudouridine in the immune system is limited. Trub1 is one of 13 pseudouridine synthases (PUS) and has been suggested to co-install the majority of pseudouridine residues on mRNA alongside the activity of PUS7 and has been reported to install pseudouridine in tRNA. Against this background, we hypothesized that Trub1 may regulate immune cell development, homeostasis or functional differentiation. Using conditional and global Trub1-deficient mice, in vivo competition in mixed bone marrow chimaeras and high-parametric flow cytometry, we interrogated the role of Trub1 in T cells, B cells, NK cells and myeloid cells. Despite our rigorous analyses and the identification of Trub1-dependent pseudouridine residues in mitochondrial tRNAs, we did not identify a significant role for Trub1-mediated pseudouridylation in immune cells at steady state or during disease. We hence reason that the role of Trub1 in mRNA pseudouridylation has previously been overestimated in computational studies or that co-installations of pseudouridine at other positions in the same RNA molecules may compensate for the lack of Trub1-installed modifications.