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| 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
Other
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| 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.
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| 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.
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| 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 |
| E-mail(s) |
igor.ulitsky@weizmann.ac.il
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| Organization name |
Weizmann Institute of Science
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| Street address |
Hertzl St.
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| City |
Rehovot |
| ZIP/Postal code |
76100 |
| Country |
Israel |
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| Platforms (2) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (141)
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| Relations |
| BioProject |
PRJNA578644 |
| SRA |
SRP226431 |
| 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 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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