EndoU, a gene within the Chr 15 locus that encodes a novel ssRNA binding protein, is overexpressed in anergic B cells. (A) Relative transcript expression of the 28 genes within the Chr 15 locus in BCR-stimulated spleen B cells from CD22^−/−[B6] (closed bars) or CD22^{−/−[inbr]} (open bars) mice. A single gene, EndoU (arrows), was differentially expressed by B cells from CD22^−/−[B6] and CD22^{−/−[inbr]} parental mice. (B) RNA was isolated from purified spleen B cells that were untreated (open bars) or were stimulated with F(ab’)₂ anti-IgM Abs for 18 h (closed bars), followed by real-time RT-PCR analysis of EndoU gene expression. Values represent mean (±SEM) fold changes in EndoU expression with Cd20 expression as the internal control gene in B cells from three mice of each genotype. All values were normalized relative to WT^[129] B cells after BCR stimulation (dashed line). The diploid genotypes of each mouse line at the Chr 15 SNP-defined locus are indicated. (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^TgsHEL mice as assessed in two independent experiments producing similar results. **, P < 0.01, Student’s t test. (E) [³²P]-labeled ssRNA substrates described for XendoU (Laneve et al., 2008) were incubated with recombinant EndoU protein in the presence or absence of Mn²⁺ 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, [³²P]-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-positive cells as determined by flow cytometry. The percent decrease in and p-value for the reduction of mean c-Myc MFI values (minus isotype control [Iso ctl] Ab staining) in EndoU-transfected cells (EndoU) relative to control-transfected cells (Vector) from four independent experiments are indicated. The significance level was determined using the Student’s t test.