Fig. 1: mEndoU is expressed during thymocyte development and has a calcium-activated RNase activity.

A mEndoU expression levels in developing thymocyte populations. The color indicates fractional expression of the mRNA across 211 measured cell types. Data from the Immunological Genome Project³². B RNase activity in WT and mEndoU KO cell lysates. Cytoplasmic lysates from the indicated WT, KO or rescue cell lines were incubated +/−5 mM calcium for 15 min at 37 °C. RNA was extracted, 5 μg were run on an 8 % urea-PAGE gel, and visualized by SYBR Green II. WT-HA denotes a WT mEndoU rescue construct with a C-terminal HA tag. C Immunoprecipitated mEndoU cleavage activity on a defined RNA substrate. D Cleavage activity of WT mEndoU (WT-HA) and the catalytically dead (CD-HA) mEndoU double mutant E285A/H286A. The initial reaction rate was significantly higher for WT-HA compared to CD-HA (p = 0.0002). E mEndoU RNase activity is specifically activated by calcium ions. The initial reaction rate was significantly higher for calcium compared to Mg²⁺ (p = 0.02), Mn²⁺ (p = 0.01), Zn²⁺ (p = 0.01), Cu2+ (p = 0.01), Pb²⁺ (p = 0.01), Co²⁺ (p = 0.01), and Ni²⁺ (p = 0.01). For (D) and (E), experiments were done in triplicate. Initial reaction rates were computed from linear regression at early time points for each replicate and they are plotted as boxplot, with the center line representing the median, the box bounds indicating the 25–75th percentiles, and the whiskers extending to the minimum and maximum values. Checks for normality and equal variance across the data were performed using the Shapiro–Wilk and Levene’s tests, respectively. Group comparisons were achieved with pairwise independent two-sample, one-sided t-tests. The p-values were adjusted using the Benjamini–Hochberg method for multiple testing correction. Source data are provided as a Source Data file. F Topology of XendoU domains across phyla. The XendoU domain (PF09412¹⁵) consists of a C-terminal catalytic core that is conserved across phyla. It features a long N-terminal extension specific to eukaryotes and a short N-terminal extension specific to nidoviruses. In eukaryotes, the presence of the N-terminal extension correlates with a dependence on metal ions.