Mapping of Nab3 RNA-binding sites in Saccharomyces cerevisiae with suboptimal expression of PIC2.

Using CRAC, we compared the transcriptomic occupancy of Nab3 in a Saccharomyces cerevisiae BY4741 parental strain (PIC2-GFP, i.e., WT) and two derived mutants overexpressing (pTEF1-PIC2) or lacking (KO) PIC2. The purpose of the experiment was to check whether overexpressing or preventing the expression of PIC2 (an established Nab3 mRNA target which, when overexpressed or deleted, causes severe cellular defects) would cause a re-distribution of Nab3 binding among its other target transcripts. 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.).本研究采用CRAC技术，对酿酒酵母（Saccharomyces cerevisiae）BY4741亲本菌株（PIC2-GFP，即野生型（Wild Type））以及两株衍生突变株的Nab3转录组结合占据率开展比较分析。上述两株突变株分别为过表达PIC2的(pTEF1-PIC2)菌株与缺失PIC2的(KO)菌株。本实验旨在验证：过表达或抑制PIC2的表达——PIC2是已被证实的Nab3 mRNA靶标，其过表达或缺失均会引发严重的细胞缺陷——是否会导致Nab3在其余靶标转录本间的结合分布发生重排。测序产出数据通过pyCRAC分析流程进行处理，结合峰调用分析则借助我们新开发的DBPeaks工具包完成；该工具包专为识别和比对由RNA结合足迹技术（如CRAC、iCLIP、PAR-CLIP等）定义的结合位点而研发。