Genome-wide screening of NEAT1 regulators reveals mito-paraspeckle communication

The long non-coding RNA NEAT1 (nuclear enriched abundant transcript 1) nucleates the formation of paraspeckles, which constitute a type of nuclear body that has multiple roles in gene expression. How the NEAT1 gene itself is regulated and how paraspeckles communicate with other cell compartments remains poorly understood. Here we identify regulators of NEAT1 transcription using an endogenous NEAT1 promoter-driven EGFP reporter in human cells coupled with genome-wide RNAi screens. In addition to transcription factors and chromatin modulators, the screens unexpectedly yielded gene candidates involved in mitochondrial functions as essential regulators of NEAT1 expression and paraspeckle formation. Mitochondrial defects altered NEAT1 transcription via ATF2 and subsequently uncoupled 3’ end processing of NEAT1_1 from its long isoform to favour NEAT1_2 production, which is key for generating elongated paraspeckles that have different features from the regular, globular bodies. Correspondingly, NEAT1 depletion has profound effects on mitochondrial dynamics and function by altering sequestration of mRNAs of mitochondrial genes enriched in paraspeckles. Overall, our data provided a rich resource for understanding NEAT1 and paraspeckle regulation, and revealed an unexpected crosstalk between cytoplasmic organelles and nuclear bodies. Examination of differential expressed genes between wild type and NEAT1 KO by poly(A)+ RNA-Seq and identifying RNAs that interact with NEAT1 by NEAT1 CHART (capture hybridization analysis of RNA targets) assays followed by RNA-sequencing (CHART RNA-seq) from HeLa cells.

长链非编码RNA NEAT1（核富集丰度转录本1，nuclear enriched abundant transcript 1）可介导旁斑（paraspeckles）的成核形成，而旁斑是一类在基因表达过程中发挥多重功能的核体（nuclear body）。目前对于NEAT1基因自身的调控机制，以及旁斑与其他细胞区室的通信方式，仍知之甚少。本研究借助人源细胞中内源性NEAT1启动子驱动的EGFP报告基因，结合全基因组RNA干扰（RNAi）筛选，鉴定出了NEAT1转录的调控因子。除转录因子与染色质调控因子外，此次筛选还意外得到了一批参与线粒体功能的基因候选物，它们是NEAT1表达及旁斑形成的关键调控因子。线粒体功能缺陷可通过ATF2通路改变NEAT1的转录，并随后解除NEAT1_1与其长异构体的3'端加工偶联，从而偏向于生成NEAT1_2，而NEAT1_2是形成伸长型旁斑的关键，此类旁斑与常规球状核体具有不同的特征。相应地，NEAT1的缺失会通过改变富集于旁斑的线粒体基因mRNA的隔离状态，对线粒体动力学及功能产生显著影响。综上，本研究的数据为解析NEAT1与旁斑的调控机制提供了丰富的资源，并揭示了细胞质细胞器与核体之间此前未被发现的交互串扰。本研究通过poly(A)+ RNA测序（poly(A)+ RNA-Seq）分析野生型与NEAT1敲除（KO）细胞间的差异表达基因，并借助海拉细胞（HeLa cells）中的NEAT1 CHART（RNA靶点捕获杂交分析，capture hybridization analysis of RNA targets）实验结合RNA测序（CHART RNA-seq），鉴定出了与NEAT1相互作用的RNA分子。