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Series GSE139151 Query DataSets for GSE139151
Status Public on May 25, 2020
Title Gene architecture and sequence composition underpin selective dependency of long RNAs on components of the nuclear export pathway
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Summary 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.
Contributor(s) Ulitsky I, Zuckerman B, Ron M
Citation(s) 32504555
Submission date Oct 21, 2019
Last update date Aug 24, 2020
Contact name Igor Ulitsky
Organization name Weizmann Institute of Science
Street address Hertzl St.
City Rehovot
ZIP/Postal code 76100
Country Israel
Platforms (2)
GPL18573 Illumina NextSeq 500 (Homo sapiens)
GPL24676 Illumina NovaSeq 6000 (Homo sapiens)
Samples (141)
GSM4132102 siALY_UAP56_cyt_rep2
GSM4132103 siALY_UAP56_cyt_rep3
GSM4132104 siALY_UAP56_nuc_rep2
BioProject PRJNA578644
SRA SRP226431

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Supplementary file Size Download File type/resource
GSE139151_Data_for_SupplementaryTable_AllSplicingRatios.xlsx 867.5 Kb (ftp)(http) XLSX
GSE139151_RAW.tar 4.2 Gb (http)(custom) TAR (of BW, TXT)
GSE139151_Table_S2_halflife.csv.gz 257.4 Kb (ftp)(http) CSV
GSE139151_finalTab_siRBM15_and_siWTAP_CytoLib.xlsx 570.0 Kb (ftp)(http) XLSX
GSE139151_nascent_RNA_expression_and_localization.csv.gz 1.5 Mb (ftp)(http) CSV
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Processed data provided as supplementary file

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