The RNA-binding profile of Acinus, a peripheral component of the Exon junction complex, reveals its role in splicing regulation. Homo sapiens

Acinus (Apoptotic Chromatin Condensation Inducer in the Nucleus) is an RNA-binding protein (RBP) originally identified for its role in apoptosis. It was later found to be an auxiliary component of the Exon Junction Complex (EJC), which is deposited at exon junctions as a consequence of pre-mRNA splicing. To uncover the cellular functions of Acinus and investigate its role in splicing, we mapped its endogenous RNA targets using individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP). We observed that Acinus binds to pre-mRNAs, associating specifically to a subset of suboptimal introns, but also to spliced mRNAs. We also confirmed the presence of Acinus as a peripheral factor of the EJC. RNA-seq was used to investigate changes in gene expression and alternative splicing following siRNA-mediated depletion of Acinus in HeLa cells. This analysis revealed that Acinus is preferentially required for the inclusion of specific alternative cassette exons and also controls the faithful splicing of a subset of introns. Moreover, a large number of splicing changes can be related to Acinus binding, suggesting a direct role of Acinus in exon and intron definition. In particular, Acinus regulates the splicing of DFFA/ICAD transcript, a major regulator of DNA fragmentation. Globally, the genome-wide identification of RNA targets of Acinus revealed its role in splicing regulation as well as its involvement in other cellular pathways, including cell cycle progression. Altogether, this study uncovers new cellular functions of an RBP transiently associated with the EJC Overall design: We performed iCLIP in triplicates in HeLa cells transfected with a T7-Acinus S* (common part of Acinus L, S and S’) or a control empty vector (pCG). Peaks present in protein coding gene regions were detected using Pyicoclip (Althammer et al, 2011) after pooling the reads from the replicates. We carried out the iCLIP-seq of endogenous Acinus-L and Acinus-S/S’ in duplicates as well as a control iCLIP after IP with rabbit IgG in HeLa cells. We performed RNA-Seq on polyA+ RNA isolated from HeLa cells following siRNA knockdown of all three Acinus isoforms. Knockdown was performed in triplicate using an Acinus siRNA together with three independent control experiments using a non-targeting siRNA.

核内凋亡染色质浓缩诱导因子（Acinus）是一种RNA结合蛋白（RNA-binding protein, RBP），最初因在细胞凋亡中的功能被鉴定。后续研究发现其为外显子连接复合体（Exon Junction Complex, EJC）的辅助组分，该复合体因前体mRNA（pre-mRNA）剪接过程沉积于外显子连接处。为解析Acinus的细胞功能并探究其在剪接中的作用，我们采用单核苷酸分辨率紫外交联免疫沉淀（individual-nucleotide resolution UV-crosslinking and immunoprecipitation, iCLIP）技术绘制了其内源RNA靶标图谱。
我们观察到，Acinus可结合前体mRNA，特异性结合一类亚最优内含子，同时也可结合剪接后的mRNA。我们还证实Acinus作为外显子连接复合体的外周因子存在。通过RNA测序（RNA-seq），我们探究了海拉细胞（HeLa）中经小干扰RNA（small interfering RNA, siRNA）介导的Acinus敲低后基因表达与可变剪接的变化。该分析显示，Acinus优先促进特定可变盒式外显子的包含，同时调控一类内含子的精确剪接。此外，大量剪接变化与Acinus的结合事件相关，提示Acinus在外显子与内含子的识别中发挥直接作用。特别地，Acinus可调控DFFA/ICAD转录本的剪接，后者是DNA片段化的主要调控因子。
全基因组范围内对Acinus RNA靶标的鉴定揭示，其不仅参与剪接调控，还参与其他细胞通路，包括细胞周期进程。综上，本研究揭示了一种暂态结合于外显子连接复合体的RNA结合蛋白的全新细胞功能。
整体实验设计：我们对转染T7-Acinus S*（Acinus L、S及S’的共通片段）的海拉细胞及转染空载体（pCG）的对照细胞进行了三次重复iCLIP实验。合并重复样本的测序reads后，我们使用Pyicoclip（Althammer等，2011）检测了蛋白编码基因区域内的结合峰。我们还对海拉细胞中内源Acinus-L及Acinus-S/S’进行了两次重复iCLIP-seq，并设置了用兔IgG进行免疫沉淀的对照iCLIP实验。我们对经针对三种Acinus亚型的小干扰RNA敲低的海拉细胞提取的poly(A)+ RNA进行了RNA测序。敲低实验设置三次生物学重复，同时使用非靶向小干扰RNA设置了三组独立对照实验。