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.

Segregation of the B cell survival versus AICD phenotypes in CD22 /[N1] mice indicated that the genomes of the parental CD22 / mouse lines were stable and amenable to genetic analyses. Therefore, a forward genetic linkage analysis was used to identify the gene locus/loci responsible for CD22 /[B6] B cell AICD. Genome-wide genotyping of 250 informative single nucleotide polymorphisms (SNPs) between the B6 and 129 mouse strains used genomic DNA from 44 CD22 /[N1] mice; 22 mice had viable B cells after BCR ligation, whereas 22 mice had B cells that underwent AICD (Fig. 2 C) . Quantitative trait loci (QTL) mapping regression analysis revealed a single locus with high logarithm of odds (LOD) scores (P < 10 6 ) on the distal end of chromosome (Chr) 15 ( Fig. 2 D) , (C) Purified spleen B cells or CD4 + T cells were cultured alone or with F(ab') 2 anti-IgM Abs or mitogenic CD3 mAb, respectively, for 18 h. Total cellular proteins were separated by SDS-PAGE, followed by transfer to nitrocellulose membranes and Western blot analysis using polyclonal anti-EndoU Ab. The arrow indicates the position of the major EndoU band, which was dominant in CD22 /[B6] B cells. A nonspecific band (n.s.) is present in all lanes that is also observed when the secondary detection Ab is used alone as a control to probe CD22 /[B6] B cell lysates (far right lane). Membranes were reprobed with a polyclonal ERK2 Ab as a control for total protein loading. Bar graphs indicate relative B cell EndoU band densities (±SEM) from three each CD22 /[B6] and CD22 /[inbr] mice assessed in independent experiments. **, P < 0.01, Student's t test. (D) Purified spleen B cells from the indicated mice were cultured alone or with F(ab') 2 anti-IgM Abs for 18 h. Total cellular proteins were separated by SDS-PAGE, followed by transfer to nitrocellulose membranes and Western blot analysis using polyclonal anti-EndoU Ab as described in C. Bar graphs indicate mean EndoU band densities from three each Ig Tg and Ig Tg sHEL mice as assessed in two independent experiments producing similar results. **, P < 0.01, Student's t test. (E) [ 32 P]-labeled ssRNA substrates described for XendoU (Laneve et al., 2008) were incubated with recombinant EndoU protein in the presence or absence of Mn 2+ or EDTA as indicated. Reactions were then separated by 15% PAGE and visualized by phosphorimager. The migration positions of EndoU-RNA complexes and of unbound RNA are indicated. Results are representative of those obtained in three experiments producing similar results. (F) c-Myc mRNA (sequence at left) shows an enrichment for poly(U) sequences. Regions of three or more consecutive uridines are highlighted. Bar graph at right shows the binding of in vitro transcribed, [ 32 P]-labeled c-Myc mRNA by recombinant EndoU protein bound to IgG-coated magnetic beads as quantified by scintillation counting. A >100-fold molar excess of nonspecific protein (BSA) and RNA (tRNAs) were used as carriers in these assays. Background c-Myc RNA binding to magnetic beads without EndoU (Carrier) is shown. Bar graphs represent means (±SEM) from three binding assays per group. Results represent one of two experiments producing similar results. (G) NIH-3T3 cells were stably transfected with a plasmid encoding EndoU fused with CFP reporter, or with plasmid expressing a reporter protein alone as a control. Shown are representative histogram overlays of immunofluorescence staining of intracellular c-Myc protein in reporter-