RNA结合蛋白PTBP3在B细胞分化发育中的调控机制研究

B lymphocytes are involved in humoral immunity, which can secrete antibodies to execute immune clearance of toxins and microorganisms. The early development and differentiation of B cells mainly occur in bone marrow, and their abnormal development is closely related to immunodeficiency and cancer. It is wellknown that RNA binding protein and non-coding RNA can regulate gene expression at post-transcriptional level to influence the development of B cell, but it is still unclear how RNA binding protein regulates B cell development at transcriptional level. We unexpectedly find that RNA binding protein PTBP3 is highly expressed in B cell, and its point mutations closely associated with inflammation and immune abnormality. PTBP3 depletion by shRNA result in B cell arrested at the Pro/Pre-B stage in bone marrow. Transcriptome analysis revealed that PTBP3 can modulate the expression of several hundred non-coding RNAs and pseudogenes. With these bases, this proposal is planning to construct stage specific knockout mice of PTBP3, and seriously analyze the regulatory mechanism of PTBP3 in transcription and metabolism of non-coding RNA during B cell development and differentiation. We hope to uncover PTBP3 mediated molecular circuits in B cell differentiation and to decipher the pathological mechanism of PTBP3 related mutations. This study will also pave the way for the clinical diagnostic of immune disorders and the treatment of B cell lymphoma.

B淋巴细胞参与体液免疫，可分泌抗体对抗病原微生物和有毒物质的侵染。B细胞早期发育和分化主要在骨髓中完成，其发育异常与免疫缺陷病和癌症发生密切相关。已知RNA结合蛋白和非编码RNA可在转录后水平调节基因表达进而影响B细胞发育，但RNA结合蛋白如何在转录水平调控B细胞分化和发育尚不明了。申请人前期研究意外发现RNA结合蛋白PTBP3在B细胞中特异性高表达，且其点突变与炎症和免疫异常疾病相关。利用shRNA对PTBP3敲降后导致B细胞发育停滞在Pro/Pre-B阶段，转录组分析发现PTBP3可调控数百个非编码RNA和假基因的表达。本申请计划在此基础上构建PTBP3发育阶段特异性敲除小鼠，深入分析PTBP3在B细胞发育分化过程中调控非编码RNA转录与代谢的机理。该研究有望解析PTBP3调控的分子环路及其突变的致病机理，为B细胞发育分化异常导致的淋巴瘤及免疫性相关疾病的临床诊治提供理论基础和新靶标。

B淋巴细胞参与体液免疫，可分泌抗体对抗病原微生物和有毒物质的侵染。B细胞早期发育和分化主要在骨髓中发生，其发育异常与免疫缺陷病和癌症发生密切相关。RNA结合蛋白和非编码RNA可在转录后水平调节基因表达进而影响B细胞发育，但RNA结合蛋白如何在转录水平调控B细胞分化和发育尚不明。本项目通过构建PTBP3发育阶段特异性敲除小鼠，深入分析PTBP3在B细胞发育分化过程中调控RNA转录与代谢的机理。借助高通量测序技术检测了PTBP3在B细胞中的转录调控方式。转录组测序结果显示，PTBP3的缺失引起了大量基因的异常表达，其中大部分基因与细胞周期相关，少部分基因与免疫应答反应和信号通路相关，可能与B1细胞发育异常相关，也有可能与B细胞中PTBP3的转录后调控相关。结合PTBP3在B细胞全基因组上的分布情况，poly(A)尾富集测序揭示了PTBP3在mRNA 3’末端加工过程中的新功能，即PTBP3可以通过特异地结合mRNA 3’UTR上CU富集（CU-rich）的序列调控poly(A)位点的选择，影响生成mRNA的3’UTR长度，而且PTBP3对可变多聚腺苷酸化（APA）事件的调控作用具有一定的位置和剂量效应。免疫共沉淀和免疫荧光技术还揭示了PTBP3通过与重要的剪切多聚腺苷酸化因子CPSF6相互作用介导不同poly(A)位点的利用。PTBP3调控的部分APA事件与mRNA的稳定性相关，影响其在初始B细胞中的丰度。结果表明PTBP3通过与CPSF6结合共同调控mRNA 3’末端poly(A)位点的选择，影响mRNA的表达，影响B细胞的生物学功能和B1细胞的发育。本项目首次发现了PTBP3在调控mRNA 3’末端加工过程中的新功能和新机制，为深入阐明PTBP3对B1细胞发育及其在天然免疫中的作用和调节机制，和APA调控治疗自身免疫疾病和免疫缺陷疾病提供了新的线索。