RNA剪切蛋白hnRPLL对淋巴细胞分化及功能的调控机制

Lymphocytes require priming and further differentiation in order to fulfill their biological functions. Lymphocyte differentiation is accompanied by the dramatic alteration of transcriptome, which is mediated by part through the post-transciptional regulation of mRNA processing, such as mRNA alternative splicing, alternative polyadenylation and mRNA stability. RNA posttranscriptional regulation is regulated stepwise through an array of RNA binding proteins with distinct functions(RBPs). The role of RBP and their mediated RNA processing events in lymphocyte differentiation however have not been appreciated until recently. More importantly perturbation of these processes in many cases is the direct cause of human genetic diseases. hnRPLL is an RNA binding protein whose expression is tightly controlled during lymphocyte activation and differentiation. In T cells, expression of hnRPLL is induced after activation and mediates splicing of CD45 from na?ve T cell CD45 isoform(CD45RA) to memory T cell isoform(CD45RO). In B cells, however, we found that hnRPLL is not induced after cell activation but rather is only expressed at plasma cell stage, suggesting an important role of this RBP in B cell to plasma cell differentiation and/or function. In order to identify hnRPLL binding sites, we conducted PAR-CLIP experiment, and found that hnRPLL could bind to more than 3000 transcripts in plasma cells. More importantly, hnRPLL was associated with a few important transcription factors for plasma cell differentiation, including XBP-1. In this proposal, we will examine the hypothesis that hnRPLL dependent post-transcriptional regulation synergizes with transcriptional regulation orchestrating differentiation and/or functions of plasma cells. We will first examine the function of hnRPLL in plasma cell differentiation through both in vitro cell culture and in vivo animal experiments; next, we will characterize the interaction between hnRPLL and its target mRNAs, focusing on Xbp-1 and CD45; Finally we will examine the cooperation between hnRPLL and its most closely related analog hnRNP L in mediating RNA splicing and stability. Results obtained through this study will not only establish hnRPLL as an important regulator of lymphocyte differentiation, but also provide mechanistic insight into interaction between RBP and their RNA substrates. Accomplishment of these studies will for the first time establish the importance of RNA binding proteins in lymphocyte lineage differentiation.

淋巴细胞的激活和分化是实现其生物学功能的重要环节，这一过程伴随着转录组的剧变，其中RNA转录后调节（包括RNA剪切和稳定性等）是转录组调控的重要步骤。RNA在转录后的命运主要取决于RNA结合蛋白(RBP)与相应的RNA序列的相互作用。因此，阐明RNA的后转录调节机制依赖于深入理解RBP和RNA的相互作用。我们发现RNA 剪切蛋白hnRPLL在B细胞中选择性的在浆细胞中表达；通过高通量测序，我们鉴定了与hnRPLL结合的RNA，发现hnRPLL和多个对浆细胞分化至关重要的转录因子的RNA相结合。本课题将以此为基础，利用体外细胞培养和动物实验，研究hnRPLL在浆细胞分化和功能中的作用；分析hnRPLL与其他剪切蛋白的相互作用；阐明hnRPLL与不同RNA序列元件的作用机制。这些研究结果将确定hnRPLL作为浆细胞分化的关键调节因子，并建立RBP作为调节淋巴细胞分化的关键分子的作用模式。

在抗原刺激激活B细胞之前，抗体分子是以膜蛋白形式表达在成熟B细胞的表面；激活的B细胞分化为终末期的浆细胞的同时，抗体也由膜型转变为分泌型分泌到体液中介导体液免疫应答。这个抗体膜型与分泌型的转变过程是由RNA结合蛋白调控的RNA选择性加工（即选择性剪接和选择性加尾两个机制）决定的。我们的研究发现在终末分化的免疫细胞中高表达的RNA结合蛋白hnRNPLL（Heterogeneous nuclear ribonucleoprotein L-like），能够调节浆细胞中免疫球蛋白重链（IgH）的膜型与分泌型的转变过程；我们还发现在T细胞和浆细胞中，RNA结合蛋白PABPC1（poly(A) binding protein, cytoplasmic 1）特异性地与hnRNPLL相互作用。虽然PABPC1并不像hnRNPLL一样，调控CD45的选择性剪接，但其能够促进浆细胞中hnRNPLL对IgH的结合并能够调节膜型与分泌型IgH的转变。考虑到目前有研究证实PABPC1参与mRNA的选择性加尾过程，我们的研究暗示PABPC1通过选择性加尾机制调控抗体的膜型与分泌型的转变，并通过与hnRNPLL相互作用促进hnRNPLL通过选择性剪接机制调控抗体的这种转变过程。我们的研究因而揭示了淋巴细胞分化过程中转录后调控的重要功能。