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. 2019 Mar 1;10:990. doi: 10.1038/s41467-019-08942-3

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — PTex is a fast method to purify cross-linked RNPs. a In vivo cross-linking of HEK293 cells using UV light at 254 nm wavelength results in covalent bonds between RNA and proteins in direct contact. Cross-linked RNPs are indicated by an orange star. b Schematic of the separation principle of biphasic organic extractions used in PTex. Left panel: Phenol-Toluol (50:50) and neutral pH results in an accumulation of proteins and RNA in the upper aqueous phase (aq) while DNA and lipids are retained at the interphase (inter). Right panel: under acidic phenol and chaotropic conditions, non-cross-linked RNA accumulates in the aqueous phase (aq), non-cross-linked proteins in the lower organic phase (org), and cross-linked RNPs (clRNPs) are enriched at the interphase (inter). c Step-by-step analysis of proteins in 9 intermediary steps of the PTex protocol (3 extractions with 3 phases each). Western blot against HuR (ELAVL1, 35 kDa) demonstrates that UV-cross-linking-stabilised HuR-RNA complexes (upper edge/gel pocket of the blot) are largely enriched after PTex (step 3 interphase). A purified fly protein (Sxl RBD4) served as spike-in as 100% non-cross-linked RBP. d 5′-end radioactive-labelled RNA was subjected to PTex in vitro. e PCR with specific primers against exon 5 of the interleukin 3 (IL3) gene demonstrates efficient removal of genomic DNA after either full HEK293 cells (upper panel) or pre-purified genomic DNA (middle panel) were subjected to PTex. A PCR product derived from linear pUC19 DNA (lower panel) is also removed. f Enrichment of known RBPs by PTex tested by western-blot against PTBP1, FUS, or against non-classical RNA-binding enzymes Eno1 and GAPDH. Note that RNaseA treatment was performed after PTex as it removes partially shifted bands (smear) for some RBPs. g PTex enriches for cross-linked RBPs. RNase treatment before PTex strongly reduces recovery of known RNA-binders (PTBP1, FUS). Non-RBP controls Histone H3 and actin (ACTB) are efficiently depleted by PTex (c, f, g). For full gels/blots see Supplementary Figures 1–8