Gene architecture and sequence composition underpin selective dependency of long RNAs on components of the nuclear export pathway

The nuclear export pathway transports long RNAs produced in the nucleus to the cytoplasm. The core components of this pathway are thought to be required for export of virtually all polyadenylated RNAs. Here, we depleted different proteins that act in nuclear export in human cells, and quantified the transcriptome-wide consequences on RNA localization. Different genes exhibited substantially variable sensitivities, with depletion of NXF1 and TREX components causing some transcripts to become strongly retained in the nucleus while others were not affected. Specifically, NXF1 is preferentially required for export of single- or few-exon transcripts with long exons or high A/U-content, whereas depletion of TREX complex components preferentially affects spliced and G/C-rich transcripts. Using massively parallel reporter assays we identified short sequence elements that render transcripts dependent on NXF1 for their export, and identified synergistic effects of splicing and NXF1. These results revise the current model of how nuclear export shapes the distribution of RNA within human cells. Overall design: In order to obtain further evidence that the observed effects are a direct consequence of NXF1 depletion, we used SLAM-seq (Herzog et al. 2017), which included labeling of newly synthesized RNA using 4-thiouridine followed by iodoacetamide treatment of extracted RNA and sequencing. We labeled RNA for 4 hours just 16 hours after transfection of siRNAs targeting NXF1, which was sufficient for partial depletion of the NXF1 protein. In order to identify sequences that may promote NXF1-dependent export of intronless RNAs, we used a massively parallel RNA assay (Lubelsky and Ulitsky 2018; Shukla et al. 2018). We designed short oligos tiled across the sequences of NORAD, ATXN7L3B, MEX3C, and eight additional single-exon, cytoplasmic, and NXF1-sensitive human genes, including six PCGs and two lncRNAs. As a control, we also included the JPX lncRNA and a fragment of the MLXIPL gene, which we studied previously (Lubelsky and Ulitsky 2018). Most of the transcripts were tiled with 140 nt sequences with offsets of 20 nt (10 nt for NORAD and 25 nt for JPX). Overall, 2,545 sequences (collectively called CytoLib) were cloned into the 3'UTR of an intronless variant of the ?-globin gene (??1,2), which is relatively inefficiently exported, and was previously used as a model sequence for study of elements affecting nuclear export (Akef, Lee, and Palazzo 2015; Brown and Steitz 2016). In order to test the potential importance of RBM15 binding and m6A in nuclear export, we knocked down RBM15 alongside its paralog RBM15B, and WTAP, a core member of the m6A writer complex, and examined localization of CytoLib tiles.

核输出通路负责将细胞核内合成的长链RNA转运至细胞质。该通路的核心组分被认为是几乎所有多腺苷酸化RNA（polyadenylated RNA）输出所必需的。本研究中，我们在人类细胞中敲降了不同的核输出相关蛋白，并定量分析了其对RNA定位的全转录组层面影响。不同基因表现出显著不同的敏感性：敲降NXF1及TREX复合物组分后，部分转录本会被强烈滞留于细胞核内，而其余转录本则不受影响。具体而言，NXF1优先参与外显子较长或A/U含量较高的单外显子/少数外显子转录本的输出；而敲降TREX复合物组分则主要影响经过剪接且G/C含量较高的转录本。本研究借助大规模平行报告基因实验（massively parallel reporter assay），鉴定出了使转录本输出依赖于NXF1的短序列元件，并揭示了剪接与NXF1之间的协同效应。上述研究结果修正了当前关于核输出如何塑造人类细胞内RNA分布的模型。

整体实验设计：为进一步验证所观察到的效应是NXF1敲降的直接结果，我们采用了SLAM-seq技术（Herzog等人，2017）：该技术先用4-硫尿苷标记新合成的RNA，随后对提取的RNA进行碘乙酰胺处理并进行测序。我们在靶向NXF1的siRNA转染16小时后，对RNA进行了4小时的标记，此时NXF1蛋白已实现部分敲降。为鉴定可能促进无内含子RNA以NXF1依赖方式输出的序列，我们采用了大规模平行RNA分析实验（Lubelsky与Ulitsky，2018；Shukla等人，2018）。我们设计了短寡核苷酸探针，对NORAD、ATXN7L3B、MEX3C以及另外8个人类单外显子、细胞质定位且对NXF1敏感的基因的序列进行平铺覆盖，其中包含6个蛋白编码基因（protein-coding genes，PCGs）与2个长非编码RNA（long non-coding RNA，lncRNA）。作为对照，我们还纳入了JPX长非编码RNA以及MLXIPL基因的一段序列片段——该片段我们此前已在研究中使用过（Lubelsky与Ulitsky，2018）。大部分转录本采用长度为140 nt（核苷酸，nucleotide）的寡核苷酸序列进行平铺覆盖，相邻序列的偏移量为20 nt（NORAD基因的覆盖序列偏移量为10 nt，JPX基因为25 nt）。最终共获得2545条序列（统称为CytoLib），将其克隆至无内含子β-珠蛋白基因（Δβ1,2）的3'非翻译区（3' untranslated region，3'UTR）中；该无内含子β-珠蛋白基因的RNA输出效率相对较低，此前曾被用作研究影响核输出的元件的模型序列（Akef、Lee与Palazzo，2015；Brown与Steitz，2016）。为验证RBM15结合与m6A修饰在核输出中的潜在重要性，我们同时敲降了RBM15及其旁系同源基因RBM15B，以及m6A甲基转移酶复合物的核心组分WTAP，并检测了CytoLib探针片段的定位情况。