Mapping of Sen1 RNA-binding sites in Saccharomyces cerevisiae with abrogated binding of Nab3 to PIC2

Using CRAC, we compared the transcriptomic occupancy of Sen1 in a Saccharomyces cerevisiae BY4741 parental strain (PIC2-GFP) and two derived mutants lacking Nab3 RNA-binding sites in PIC2. The purpose of the experiment was two-fold: on the one hand, we aimed to verify that the mutations inserted in the Nab3 binding sequences of PIC2 affected the binding of Sen1 to the PIC2 transcript; on the other hand, we wanted to check whether differential binding of Nab3 to PIC2 would affect how the protein bound other targets in the genome. Sequencing outputs were processed using the pyCRAC pipeline and peak calling was performed with DBPeaks, our newly developed package for identification and comparison of binding sites defined by RNA-binding footprinting techniques (e.g., CRAC, iCLIP, PAR-CLIP, etc.).本研究利用CRAC技术（Cross-Linking and Analysis of cDNAs），对比了酿酒酵母（Saccharomyces cerevisiae）BY4741亲本菌株（PIC2-GFP）以及两株缺失PIC2区域内Nab3 RNA结合位点的衍生突变株中Sen1的转录组结合占有率。本实验的研究目标分为两点：其一，验证在PIC2的Nab3结合序列中引入的突变是否会影响Sen1与PIC2转录本的结合；其二，探究Nab3与PIC2的差异性结合是否会影响该蛋白与基因组内其他靶标位点的结合模式。测序产出数据通过pyCRAC分析流程进行处理，并借助本团队新开发的结合位点鉴定与对比软件包DBPeaks完成峰识别分析；该软件包可用于鉴定和比对由RNA结合足迹技术（RNA-binding footprinting techniques，如CRAC、iCLIP、PAR-CLIP等）所定义的结合位点。