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.

EndoU function in mammals is unknown, although EndoU transcripts are abundant in glucocorticoid-sensitive thymocytes undergoing apoptosis (Baughman et al., 1992) . Highly conserved EndoU orthologs are found in many advanced and primitive species (see Fig. 8 A; Renzi et al., 2006) , but EndoU paralogs derived from ancestral gene duplications do not exist in mice. The Xenopus ortholog of EndoU, XendoU, has been defined as an endonuclease targeting ssRNA molecules harboring polyuridine (poly(U)) regions (Renzi et al., 2006) . Because EndoU substrates have not been defined in other species and EndoU-related molecules that could share redundant activities are not found in mice, the ability of EndoU to bind and/or process XendoU ssRNA substrates was examined. Recombinant EndoU protein specifically bound poly(U) ssRNAs, but not ssRNA species lacking these sites ( Fig. 3 E) . However, EndoU did not cleave these poly(U) ssRNAs, even A targeting vector was generated that contained a neomycin-resistance gene (neo r ) introduced into two unique BsmBI restriction sites, which introduced an in-frame termination codon. A thymidine kinase gene (tk) was inserted at the 5 end of the EndoU gene sequence. (B) Homologous recombination of the targeting vector into the EndoU gene of heterozygous and homozygous offspring expanded the endogenous 7.5 kb HindIII digested genomic DNA fragment into an 8.5 kb fragment as assessed by Southern blot. (C) Appropriate targeting of the EndoU gene was further confirmed by PCR analysis (primer locations shown in A), whereby a common forward primer (PCR1) was used in combination with reverse primers either adjacent to (PCR2) or within (PCR3) the neo r gene. Results show amplification of genomic DNA from WT, heterozygous, and homozygous offspring. (D) Western blot analysis of thymocyte lysates confirmed the absence of EndoU protein in EndoU / mice. The presence of a nonspecific band (n.s.) as described in Fig. 3 ) . The presence of a nonspecific band (n.s.) also described above is indicated. (F) The difference in EndoU protein migration results from a nonsynonymous SNP in the second exon of the EndoU gene. EndoU protein of CD22 /[inbr] and A/J mice are identical in sequence, but different from that of CD22 /[B6] mice as indicated by nucleotide sequence shown and protein migration by SDS-PAGE. The presence of a nonspecific band (n.s.) as also described above is indicated.