【作者】 李洋；

【导师】 鲁志（Zhi John Lu）；

【作者基本信息】 清华大学 ， 生物学， 2018， 博士

【摘要】 RNA结合蛋白(RNA-binding protein,RBP)对于细胞维持如剪接,聚腺苷酸化,RNA转运,翻译和转录本降解等一系列基本的细胞学功能至关重要。基于一项研究工作的估计,人类基因组中存在超过1500种不同的RBP,这些RBP通过结合不同的RNA靶标序列,进而行使其相应的生物学功能。许多RNA结合蛋白在结合其RNA靶标时存在相互作用或竞争的关系,因此,研究RNA结合蛋白的聚类组合方式和鉴定相应的RNA调控元件,对研究各种后转录调控机制至关重要。近几年来,紫外交联免疫沉淀结合高通量测序(High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation,CLIP-seq)技术的出现,使得研究人员能够在不同哺乳动物细胞中鉴定转录组范围内的、具有高分辨率的RNA-RBP的结合位点。目前,这些RBP的结合位点已经被很好地整理和收录在诸如CLIPdb,POSTAR和STARbase等重要的数据库中,为了揭示重要的后转录调控机制,如今越来越多的CLIP-seq数据被产出,发展整合多套CLIP-seq数据的方法,并对RBP相互作用进行系统评估,则显得愈发关键。我们收集了327套来自于HEK293/HEK293T、HepG2、K562三种细胞系,PAR-CLIP,HITS-CLIP和eCLIP三种不同技术方法的CLIP-seq数据。由于CLIPseq数据的异质性现象严重,我们建立了一套统一的重叠过滤策略,并成功鉴定得到了约45万个可靠的RBP结合峰。由于传统的层次聚类并不能够很好的解决RBP之间的关系,我们通过利用非负矩阵分解这种软聚类的方法,鉴定得到了一系列具有已知物理相互作用和免疫共沉淀实验支持的RBP聚类组合,并且定义了与这些聚类组合相关的RBP结合位点。利用这些结合位点,我们不但可以富集出已知的或新型的RNA motif,同时也证明了它们潜在的与RNA降解、RNA剪切等生物学功能密切相关,可能是真实存在的调控元件。我们的数据整合分析方法,可以广泛的应用到其他数据集上,并且可以克服由样品和实验技术异质性带来的影响。为了方便研究者对这些RBP聚类组合与结合位点的验证,我们建立了一个网页版的平台,以便分享我们鉴定的结果和原始代码。更多还原

【Abstract】 RNA-binding proteins(RBPs)are essential to sustain fundamental cellular functions,such as splicing,polyadenylation,transport,translation,and degradation of RNA transcripts.One study estimated that more than 1500 different RBPs exist in human.These RBPs cooperate or compete with each other in binding their RNA targets.Many RBPs are capable of binding different RNA targets,partially by associating with different co-factors.At the same time,some consensus RNA sequence motifs are recognized by homologous RBPs or homologous domains.Thus,proteins and RNAs appear to interact in a combinatorial manner.Crosslinking immunoprecipitation sequencing(CLIP-seq)technologies have enabled researchers to characterize transcriptome-wide binding sites of RNA-binding protein with high resolution.The CLIP-seq data of multiple RNA binding proteins have been curated and annotated in specific databases,such as CLIPdb,POSTAR,and STARbase.Several significant studies improved the prediction of individual RBPs’ binding sites by training on CLIP-seq and RNAcompete datasets.Systematic assessment of combinatory regulation of multiple RBPs would be more beneficial to derive precious biological information from various high-throughput CLIP-seq data.We collected 327 CLIP-seq datasets in three cell lines generated from three technical approaches: PAR-CLIP,HITS-CLIP,and eCLIP.Deposited CLIP-seq datasets display significant variety.We apply a soft-clustering method,RBPgroup,to various CLIP-seq datasets to group together RBPs that specifically bind the same RNA sites.We provide a unified and high-confidence set of protein-binding RNA sites and clustered RBP groups,which were validated by the known physical interactions and co-IP experiments.The binding sites defined by our method were more enriched with known motifs and better correlated with RNA degradation data and alternative splicing data than the binding peaks of single RBPs.In summary,we show that integrating public CLIP-seq datasets can provide novel insights into the combinatorial classification of RBPs.We shared our method and code,as well as the derived RNA regulatory elements,with the RNA community via a web-based platform.更多还原