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. 2019 May 1;33(9-10):482–497. doi: 10.1101/gad.319590.118

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© 2019 Palangat et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 2. — U2AF1 binds RNA in the cytoplasm. (A) Single molecule FISH analysis of IL8 mRNA expression in wt/wt, wt/S34F mutant cells plotted as frequency histogram of IL8 mRNA per cell. (Inset) Single molecule RNAFISH image (gray) showing heterogeneity in IL8 RNA expression and nuclei counter stained with DAPI (blue) in wt/wt cells. (B) Quantitative immunofluorescence detection of cytoplasmic U2AF1 (gray) in PFA fixed cells showing heterogeneity in U2AF1 distribution in the cytoplasm and nuclei counterstained with DAPI (blue). (C) Box plot (min, max, first and third quartiles, and median) showing the fraction of U2AF1 in the nucleus and cytoplasm of individual wt/wt and wt/S34F cells. (D) Western blot analysis showing presence of U2AF1 in the cytosolic fraction. RBM10 and RPA32 (increasing amounts) served as controls for any nuclear protein contamination in the cytoplasmic fraction. (E) U2AF1 inteacts with mature IL8 mRNA in the cytoplasm. RNA isolated from immunoprecipitates of cytoplasmic U2AF1 (B) was analyzed by RT-qPCR for association of IL8, IL1α, DNA polH, and RPB3 mRNA. Fraction of IL1α, IL8, DNA polH, and RPB3 mRNA bound to U2AF1 (top) and their relative enrichment to U2AF1 in mutant cells (bottom). (F) Transcriptome-wide analysis of WT U2AF1 bound cytoplasmic mature polyadenylated mRNA by RIP-seq. Scatter plot of enrichment of U2AF1 bound RNA (gray), intron-less Histone RNA (red) and mTOR regulated RNA (blue). (G) Box plot analysis of mRNA enriched in RIP over input or control IgG in the top decile of RIP, mTOR sensitive mRNA, EIF4A sensitive mRNA, or background mRNA. (H) 3′ splice site-like “AG” sequence and polypyrimidine-rich TOP motifs in 5′-UTRs of mTOR-regulated mRNAs bound by cytoplasmic U2AF1. Gene ontology terms of wild-type U2AF1-enriched cytoplasmic mRNA.