Polypyrimidine tract binding proteins are essential for B cell development

During B cell development, recombination of immunoglobulin loci is tightly coordinated with the cell cycle to avoid unwanted rearrangements of other genomic locations. Several factors have been identified that suppress proliferation in late-pre-B cells to allow light chain recombination. By comparison, our knowledge of factors limiting proliferation during heavy chain recombination at the pro-B cell stage is very limited. Here we identify an essential role for the RNA-binding protein Polypyrimidine Tract Binding Protein 1 (PTBP1) in B cell development. Absence of PTBP1 and the paralog PTBP2 results in a complete block in development at the pro-B cell stage. PTBP1 promotes the fidelity of the transcriptome in pro-B cells. In particular, PTBP1 controls a cell cycle mRNA regulon, suppresses entry into S-phase and promotes progression into mitosis. Our results highlight the importance of S-phase entry suppression and post-transcriptional gene expression control by PTBP1 in pro-B cells for proper B cell development. mRNAseq libraries were prepared from FrB pro-B cells. FrB pro-B cells were isolated from the bone marrow by flow cytometry and cell sorting of B220+, CD19+, IgD-, IgM-, CD2-, CD25-, CD43high, cKIT+, CD24+ and CD249- cells. Bone marrow cells from 4-6 mice from the same genotype and sex were pooled and depleted of unwanted cells with anti Gr-1-bio (RB6-8C5), CD11b (M1/70), IgD-bio (11-26c.2a), NK1.1-bio, CD3e (145-2C11) and Ter119 biotinylated antibodies and anti-biotin microbeads before cell sorting. Five biological replicates were carried out per genotype. iCLIP libraries were described in Monzon-Casanova, E. et al. The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers. Nat. Immunol. 45, 471 (2018).