Genome-wide Identification of DEAD-box RNA Helicase Targets Reveals Roles for RNA Secondary Structure Remodeling in mRNA Processing (iCLIP-seq)

Accurate gene expression requires the coordination of RNA processing with assembly of messenger RNA-protein (mRNP) complex. RNA helicases are a class of enzymes that unwind RNA duplexes in vitro and have been are proposed to remodel RNA structure in vivo. Herein, we provide evidence that the DEAD-box protein Dbp2 remodels RNA structure to facilitate efficient pre-mRNA processing in S. cerevisiae. First, we find that Dbp2 associates with the 3’ ends and 3’ splice-sites of mRNAs genome-wide. Using structure-seq to map RNA secondary structure, we find altered secondary structures in dbp2∆ cells that overlap polyadenylation elements and correlate with inefficient termination. We also identify a role for Dbp2 in pre-mRNA splicing and show that both splicing and termination require Dbp2 helicase activity. This reveals that DEAD-box RNA helicases unwind structure in vivo and that structural alteration of pre-mRNA is essential for proper gene expression. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) of Dbp2 and Set1 in Saccharomyces cerevisiae. 3 biological replicates for both Dbp2- and Set1-iCLIP. Set1-iCLIP serves as a negative control in this study.

精准的基因表达需要RNA加工与信使RNA-蛋白质复合物（messenger RNA-protein, mRNP）的组装协同进行。RNA解旋酶是一类可在体外解开RNA双链的酶，且被认为可在体内重塑RNA结构。本研究中，我们提供证据表明：DEAD-box蛋白Dbp2可重塑RNA结构，以促进酿酒酵母（Saccharomyces cerevisiae, S. cerevisiae）中高效的前体mRNA加工过程。首先，我们发现Dbp2在全基因组范围内结合mRNA的3'末端与3'剪接位点。我们利用结构测序（structure-seq）绘制RNA二级结构图谱，发现dbp2∆细胞中存在异常的RNA二级结构，这些结构与多聚腺苷酸化元件重叠，且与低效的转录终止过程相关。我们还确定了Dbp2在前体mRNA剪接中的功能，并证实剪接与转录终止过程均依赖于Dbp2的解旋酶活性。该研究揭示：DEAD-box RNA解旋酶可在体内解开RNA结构，且前体mRNA的结构重塑对于正常基因表达至关重要。本研究针对酿酒酵母（Saccharomyces cerevisiae）中的Dbp2与Set1，开展了单核苷酸分辨率紫外交联免疫沉淀（Individual-nucleotide resolution UV crosslinking and immunoprecipitation, iCLIP）实验。该实验中，Dbp2-iCLIP与Set1-iCLIP均设置3次生物学重复；其中Set1-iCLIP在本研究中作为阴性对照。