Deciphering the functions of human exon junction complexes

剪接不仅是大多数哺乳动物前mrna成熟的必要步骤，而且还会在mrna上沉积外显子连接复合物（EJC）。EJC调控基因表达的几个转录后步骤，包括mRNA定位、翻译和周转。此外，缺乏EJC成分的细胞由于未知的机制导致剪接发生显著变化。我们最近发现EJCs有效地抑制了附近外显子剪接位点（SS）的剪接。因此，EJC招募剪接调节因子RNPS1来抑制隐型5’SS的使用，并通过重剪接（也称为递归剪接）防止外显子跳变。此外，EJC核心的沉积直接掩盖了重组的3' SS，从而阻止了转录本的解体。因此，EJCs保护哺乳动物细胞的转录组免受无意中外显子序列的丢失，从而保护完整的全长mrna的表达。在我们的实验分析中，我们发现ejc依赖性剪接变化涉及至少三个额外的因素- RNPS1， SAP18和PNN。然而，高质量的RNA-Seq数据存在于RNPS1，但没有SAP18， PNN或EJC核心因子（EIF4A3, RBM8A, MAGOHA&B）。因此，我们计划对这些因子缺失的细胞以及cas3敲除细胞系进行RNA-Seq实验。传统Illumina测序将在纳米孔平台（Oxford nanopore Technologies）上与单分子RNA测序/单分子PCR cDNA测序相结合。此外，我们希望分别使用转录组范围的SLAM-Seq和Ribo-Seq来了解CASC3对mRNA稳定性和mRNA翻译的重要性。这些技术的结合将使我们能够更好地理解EJC的各种细胞功能。

Splicing is not only an essential step for the maturation of most mammalian pre-mRNAs but also deposits exon junction complexes (EJC) on mRNAs. The EJC regulates several post transcriptional steps of gene expression, including mRNA localization, translation and turnover. Moreover, cells lacking EJC components display remarkable changes in splicing caused by unknown mechanisms. We have recently discovered that EJCs efficiently suppress splicing of nearby exonic splice sites (SS). Hereby, the EJC recruits the splicing regulator RNPS1 to inhibit cryptic 5' SS usage and to prevent exon skipping by re-splicing (also known as recursive splicing). In addition, the deposition of the EJC core directly masks reconstituted 3' SS and thereby precludes transcript disintegration. Thus, EJCs protect the transcriptome of mammalian cells from inadvertent loss of exonic sequences and thereby safeguard the expression of intact, full length mRNAs.During our experimental analysis we discovered that EJC-dependent splicing changes involve at least three additional factors - RNPS1, SAP18 and PNN. However, high-quality RNA-Seq data exist for RNPS1, but not for SAP18, PNN or the EJC core factors (EIF4A3, RBM8A, MAGOHA&B). Therefore, we plan to carry out RNA-Seq experiments with cells, in which these factors have been depleted, as well as a CASC3 knockout cell line. Conventional Illumina sequencing will be combined with single molecule RNA sequencing/single molecule PCR cDNA sequencing on the nanopore platform (Oxford Nanopore Technologies). In addition, we want to understand the importance of CASC3 on mRNA stability and mRNA translation using transcriptome-wide SLAM-Seq and Ribo-Seq, respectively. The combination of these techniques will enable us to better understand the various cellular functions of the EJC.