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. 2020 Jan 15;11:293. doi: 10.1038/s41467-019-14109-x

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© The Author(s) 2020

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. 4 — a In vitro cleavage assay with HeLa nuclear extract (NE) using L3 (top panel) or ICP27 (lower panel) PAS pre-mRNA substrate. 5′ cleavage product is marked. Different amounts of recombinant MBP-ICP27 (labeled as ICP27) or MBP-MS2 (Ctrl) were added to NE. b Dual luciferase reporter assay. The top panel is a diagram for pPASPORT vector and the site where the PAS to be tested is inserted is marked as PAS (in a red box). Seven distinct PASs were cloned into pPASPORT, co-transfected with a vector or ICP27-expression plasmid. The Rluc/Fluc ratio, which measures cleavage/polyadenylation efficiency at the test PAS, are plotted as mean ± s.d. c pPASPORT assays with L3 and ICP27 PAS. Empty vector, ICP27 or its various mutants were co-expressed with the pPASPORT constructs and renilla/firefly luciferase ratio was plotted as mean ± s.d. d Empty vector, ICP27 or its various mutants were transiently expressed and transcription read-through at the SRSF3 and DDX5 genes were measured by qRT-PCR and plotted as mean ± s.d. e In vitro cleavage/polyadenylation reactions (similar to a) were resolved on a native gel and visualized by phosphorimaging. The mRNA 3′ processing complex (P complex) and the heterogeneous complex (H complex) are marked. f 3MS2-L3 or 3MS2-ICP27 PAS RNA substrates are incubated under cleavage/polyadenylation conditions and the assembled RNP complexes were pulled down by using amylose beads and analyzed by western blotting. Source data are provided as a Source Data file.