Polypyrimidine tract binding proteins are essential for B cell development. 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. Overall design: 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).

在B细胞发育进程中，免疫球蛋白基因座的重组与细胞周期紧密协同，以避免其他基因组区域发生异常重排。目前已鉴定出多种可抑制晚期前B细胞增殖的因子，保障轻链重组顺利进行。相较之下，我们对祖B细胞（pro-B cell）阶段重链重组过程中限制细胞增殖的因子的认知仍极为有限。
本研究鉴定出RNA结合蛋白多嘧啶束结合蛋白1（Polypyrimidine Tract Binding Protein 1, PTBP1）在B细胞发育中的关键调控作用。PTBP1及其旁系同源蛋白PTBP2的缺失会导致B细胞发育完全阻滞于祖B细胞阶段。
PTBP1可维持祖B细胞的转录组保真度。具体而言，PTBP1调控一套细胞周期mRNA调控子，抑制细胞进入S期并促进有丝分裂进程。
本研究结果凸显了PTBP1在祖B细胞中抑制S期进入以及转录后基因表达调控对于B细胞正常发育的重要性。
整体实验设计：从FrB型祖B细胞中制备mRNA测序文库。FrB型祖B细胞通过流式细胞术与细胞分选从骨髓中分离获得，分选标记为B220+、CD19+、IgD-、IgM-、CD2-、CD25-、CD43高表达（CD43high）、cKIT+、CD24+及CD249-。
将4-6只同基因型、同性别的小鼠骨髓细胞混合，使用抗Gr-1-bio（RB6-8C5）、CD11b（M1/70）、IgD-bio（11-26c.2a）、NK1.1-bio、CD3e（145-2C11）及Ter119生物素化抗体，搭配抗生物素微磁珠去除无关细胞后，再进行细胞分选。每种基因型设置5个生物学重复。
iCLIP文库的构建方法已在Monzon-Casanova, E. 等人的研究中发表：《RNA结合蛋白PTBP1是生发中心B细胞选择所必需的》，《自然·免疫学》（Nat. Immunol.）45, 471 (2018)。