Essential requirement for polypyrimidine tract binding proteins 1 and 3 in the maturation and maintenance of mature B cells in mice

The maturation of immature B cells and the survival of mature B cells is stringently controlled to maintain a diverse repertoire of antibody specificities while avoiding self-reactivity. At the molecular level this is regulated by signalling from membrane immunoglobulin and the BAFF-receptor which sustain a pro-survival programme of gene expression. Whether and how posttranscriptional mechanisms contribute to B cell maturation and survival remains poorly understood. Here we show that the polypyrimidine tract binding proteins (PTBP) PTBP1 and PTBP3 bind to a large and overlapping set of transcripts in B cells. Both PTBP1 and PTBP3 bind to introns and exons where they are predicted to regulate alternative splicing. Moreover, they also show high density of binding in the 3' untranslated regions suggesting they influence the transcriptome in diverse ways. We show that PTBP1 and PTBP3 are required in B cells beyond the immature cell stage to sustain transitional B cells and the B1, marginal zone and follicular B cell lineages. Therefore, PTBP1 and PTBP3 promote the survival of quiescent B cells by regulating gene expression at the post-transcriptional level. Overall design: PTBP3 iCLIP from in vitro activated B cells (LPS 48h). Three libraries were prepared from the same iCLIP sample where the membrane containing the RNA-RBP complexes was cut at three different molecular weight ranges

未成熟B细胞的成熟过程与成熟B细胞的存活受到严格调控，以维持多样化的抗体特异性库，同时规避自身反应性。在分子层面，该调控由膜免疫球蛋白（membrane immunoglobulin）与B细胞活化因子受体（BAFF-receptor）介导的信号通路实现，二者可维持促存活的基因表达程序。目前，转录后机制是否以及如何参与B细胞成熟与存活过程，仍知之甚少。
本研究证实，多嘧啶束结合蛋白（polypyrimidine tract binding proteins, PTBP）家族中的PTBP1与PTBP3可结合B细胞内一套庞大且存在重叠的转录本。PTBP1与PTBP3均能结合内含子与外显子，据此预测二者可调控可变剪接；此外，二者在3'非翻译区（3' untranslated regions）也呈现高密度结合特征，提示它们可通过多种途径影响转录组。
我们发现，在B细胞中，PTBP1与PTBP3的功能需求不限于未成熟细胞阶段，二者对维持过渡态B细胞以及B1、边缘区与滤泡B细胞谱系至关重要。因此，PTBP1与PTBP3可通过转录后水平调控基因表达，促进静息B细胞的存活。
整体实验设计：对体外活化的B细胞（经脂多糖（LPS）处理48小时）进行PTBP3紫外交联免疫沉淀测序（iCLIP）。从同一iCLIP样本中制备了三个文库，该样本中包含RNA-核糖核蛋白复合物的膜被切割为三种不同的分子量范围。