Department of Physics, The Ohio State University, Columbus, Ohio 43210, USA.
Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA.
RNA. 2020 Sep;26(9):1216-1233. doi: 10.1261/rna.074856.120. Epub 2020 May 28.
In eukaryotic cells, proteins that associate with RNA regulate its activity to control cellular function. To fully illuminate the basis of RNA function, it is essential to identify such RNA-associated proteins, their mode of action on RNA, and their preferred RNA targets and binding sites. By analyzing catalogs of human RNA-associated proteins defined by ultraviolet light (UV)-dependent and -independent approaches, we classify these proteins into two major groups: (i) the widely recognized RNA binding proteins (RBPs), which bind RNA directly and UV-crosslink efficiently to RNA, and (ii) a new group of RBP-associated factors (RAFs), which bind RNA indirectly via RBPs and UV-crosslink poorly to RNA. As the UV crosslinking and immunoprecipitation followed by sequencing (CLIP-seq) approach will be unsuitable to identify binding sites of RAFs, we show that formaldehyde crosslinking stabilizes RAFs within ribonucleoproteins to allow for their immunoprecipitation under stringent conditions. Using an RBP (CASC3) and an RAF (RNPS1) within the exon junction complex (EJC) as examples, we show that formaldehyde crosslinking combined with RNA immunoprecipitation in tandem followed by sequencing (xRIPiT-seq) far exceeds CLIP-seq to identify binding sites of RNPS1. xRIPiT-seq reveals that RNPS1 occupancy is increased on exons immediately upstream of strong recursively spliced exons, which depend on the EJC for their inclusion.
在真核细胞中，与 RNA 结合的蛋白质可以调节其活性，从而控制细胞功能。为了充分阐明 RNA 功能的基础，必须鉴定出这些与 RNA 相关的蛋白质、它们在 RNA 上的作用方式以及它们首选的 RNA 靶标和结合位点。通过分析紫外线（UV）依赖和非依赖方法定义的人类 RNA 相关蛋白目录，我们将这些蛋白分为两大类：（i）广泛认可的 RNA 结合蛋白（RBPs），它们直接结合 RNA，并且与 RNA 高效 UV 交联；（ii）一组新的 RBP 相关因子（RAFs），它们通过 RBPs 间接结合 RNA，并且与 RNA 的 UV 交联效率较低。由于 UV 交联和免疫沉淀随后测序（CLIP-seq）方法不适用于鉴定 RAFs 的结合位点，我们表明甲醛交联稳定 RAFs 在核糖核蛋白内，以允许在严格条件下进行免疫沉淀。我们以 RNA 剪接体复合物（EJC）中的 RBP（CASC3）和 RAF（RNPS1）为例，表明甲醛交联与 RNA 免疫沉淀串联进行随后测序（xRIPiT-seq）大大优于 CLIP-seq，以鉴定 RNPS1 的结合位点。xRIPiT-seq 揭示了 RNPS1 占据了强递归剪接外显子上游的外显子，这些外显子依赖 EJC 进行包含。
