A Bacterial Artificial Chromosome system to uncover RNA Binding Protein functions in deep intronic regions [eCLIP]

The processing of long introns in alternative splicing of pre-mRNAs is still not well understood. Using bacterial artificial chromosome (BAC) recombineering technology, we generated a Ntrk2-BAC minigene including endogenous 50 Kb introns flanking alternatively expressed exons generating two isoforms. We found that the RNA-binding protein RbFox1 regulates the expression of the minigene-encoded isoforms. Enhanced RNA Cross-Linking Immuno-Precipitation (eCLIP), revealed that RbFox1 binds clusters of (U)GCAUG motifs present in deep intronic regions. Deletion of these clusters completely abolished RbFox1-mediated Ntrk2-isoform expression regulation. Bioinformatic analysis showed (U)GCAUG-clusters are present in both mouse and human genes. Integrative data analysis from eCLIP and RNAseq experiments showed a global increase in RNA isoform modulation of genes with Rbfox1 eCLIP-peaks associated with these clusters. These data suggest that BACs are powerful tools to study RNA metabolism across large genomic fragments and clustered distal intronic Rbfox motifs are important determinants of RbFox1 function in the mammalian genome providing a target for identification of intronic pathogenic mutations. eCLIP was performed using the 77-5 clonal cell line stably expressing the Ntrk2-BAC minigene and the 32-1 cell line stably expressing the Ntrk2-BAC minigene with Cluster 1 and Cluster 2 deletion. In the eCLIP experiment, two samples (replicates) of 77-5 cells without any treatment were used as control while two samples of 77-5 cells and two samples of 32-1 cells (replicates) were treated with Doxycycline 0.5 mg/ml for 48 hours in order to induce Rbfox1 expression in the cells (tetracycline inducible system).

前体mRNA可变剪接过程中长内含子的加工机制仍未被充分阐明。本研究借助细菌人工染色体（bacterial artificial chromosome, BAC）重组工程技术，构建了包含侧翼连接内源50 kb内含子的可变剪接外显子的Ntrk2-BAC迷你基因，该基因可产生两种剪接异构体。研究发现RNA结合蛋白RbFox1可调控该迷你基因编码的剪接异构体的表达。增强型RNA交联免疫沉淀（enhanced RNA Cross-Linking Immuno-Precipitation, eCLIP）实验结果显示，RbFox1可结合位于内含子深层区域的(U)GCAUG基序簇。删除这些基序簇后，RbFox1介导的Ntrk2剪接异构体表达调控作用完全消失。生物信息学分析表明，(U)GCAUG基序簇广泛存在于小鼠和人类的基因中。结合eCLIP与RNA测序（RNA sequencing, RNA-seq）数据的整合分析显示，携带与这些基序簇相关的Rbfox1 eCLIP结合峰的基因，其RNA剪接异构体的全局调控水平显著上调。上述研究结果表明，细菌人工染色体是研究大片段基因组片段中RNA代谢过程的有力工具；而远端内含子区域成簇分布的Rbfox结合基序，是哺乳动物基因组中RbFox1发挥功能的重要调控元件，可为内含子区域致病突变的鉴定提供潜在靶点。本研究使用稳定表达Ntrk2-BAC迷你基因的77-5克隆细胞系，以及稳定表达携带Cluster 1与Cluster 2缺失的Ntrk2-BAC迷你基因的32-1细胞系开展eCLIP实验。本次eCLIP实验中，以未接受任何处理的77-5细胞的两份重复样本作为对照；同时将两份重复的77-5细胞与两份重复的32-1细胞置于0.5 mg/ml多西环素中培养48小时，通过四环素诱导系统激活细胞内Rbfox1的表达。