EndoU is a novel regulator of AICD during peripheral B cell selection

Poe, Jonathan C.; Kountikov, Evgueni I.; Lykken, Jacquelyn M.; Natarajan, Abirami; Marchuk, Douglas A.; Tedder, Thomas F.

up-regulation is uniquely impaired in CD22 /[B6] B cells (Poe et al., 2004) , c-Myc expression was normalized in CD22 /[B6] B cells by EndoU deficiency, EndoU bound c-Myc mRNA in vitro, and ectopic EndoU expression in a fibroblast cell line reduced c-Myc levels. A regulatory role for c-Myc in the mouse WEHI-231 B cell model of AICD has been previously described (Sonenshein, 1997; Donjerković and Scott, 2000) . In CD22 / mice, EndoU overexpression and AICD required B6 homozygosity at the EndoU locus. Therefore, genetic alterations must exist within the EndoU locus between the B6 and 129 genetic backgrounds that positively influences EndoU [B6] allele transcription in B cells, but not T cells. Although the EndoU locus has yet to be finemapped or sequenced in 129 mice, these important regulatory elements are likely to be located within the numerous stretches of conserved noncoding DNA present within the Figure 7 . sHEL auto-Ag consumption occurs in high-responder EndoU / Ig Tg sHEL mice. (A) Blood cells from the indicated genotypes were stained with fluorescently labeled HEL protein, and with IgM a and B220 Abs. Top panels show dot plots for HEL and B220 staining in the lymphocyte gate, with bottom panels representing the cells in the top panels with additional gating on B220 + cells only. Results are representative of three mice of each genotype producing similar results. (B) Purified spleen B cells from high-responder EndoU / Ig Tg sHEL mice were labeled with cell tracking dye and adoptively transferred into the peritoneal cavities of either WT or sHEL recipient mice. After 36 h, peritoneal cavity cells were harvested from recipient mice and stained with fluorescently labeled HEL protein or IgM a Abs. Results represent one of two independent adoptive transfers producing similar results. (C) Histogram overlays for IgM a expression and HEL-binding levels from the B220 + donor cell populations shown in B. Solid black line, donor cells recovered from the WT recipient mouse; solid gray line, donor cells recovered from the sHEL recipient mouse; dashed line, background staining of B220 + cells in the WT recipient mouse. Results represent one of two independent adoptive transfers producing similar results. EndoU transcripts expressed in mouse B cells encoded a protein of 454 amino acids, which includes an N-terminal somatomedin B-like domain believed to be involved in protein-protein interactions (Gijsbers et al., 2003) , a XendoU Superfamily ssRNA-binding domain, and a predicted transmembrane domain. EndoU specifically interacted with an ssRNA substrate harboring U-rich sequences that is also bound by Xen-doU, which is implicated in small nucleolar RNA processing in Xenopus (Gioia et al., 2005; Renzi et al., 2006) . XendoU apparently generates snoRNAs through the cleavage of pre-mRNAs encoded within introns (Laneve et al., 2003) . Human EndoU may exhibit low-level U-directed ssRNA endoribonuclease activity against the same in vitro ssRNA substrates recognized by XendoU (Laneve et al., 2008) . However, mouse EndoU did not demonstrate measurable endonuclease activity against XendoU substrates. Mammalian EndoU may thus be functionally distinct from its Xenopus ortholog because human EndoU is excluded from the nucleolus of multiple cell lines (http://www.proteinatlas.org/ENSG00000111405/subcellular).