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. 2022 Jul 7;41(33):3979–3990. doi: 10.1038/s41388-022-02386-8

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© The Author(s) 2022

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. 7 — A CircME1 was abundantly distributed both in nucleus and cytoplasm as demonstrated by RNA-FISH with CY3-labeled circME1 probe and DAPI labeled nuclei. B, C U1-70K, U1-A, Sm-D2 and Sm-E (protein components of U1 snRNP) were identified as circME1-interacting proteins by RNA pull-down assay. The proteins pulled down by circME1 or CTRL probes were subject to SDS-PAGE and silver staining (B). U1-70K and U1-A were pulled down by circME1 as demonstrated by Western blot analysis (C). D CircME1 was markedly enriched in U1-A-immunoprecipitated RNA as demonstrated by RIP assay with IgG as negative control (left panel). Electrophoresis analysis result of qPCR products of immunoprecipitated RNA is shown (right panel). E U1-A bound to ME1 promoter (around −500 bp–−300 bp) in 786-O cells as demonstrated by ChIP assay. Knockdown of circME1 decreased the enrichment level of this DNA sequence (left panel). Electrophoresis analysis result of the ChIP products is shown (right panel). F The schematic diagram of cis-regulatory effect of circME1 on its parental gene. G, H qPCR and Western blot analyses showing that overexpression of circME1 containing mutant binding site of U1 snRNP failed to increase the expression level of ME1. I CircME1 containing mutant binding site of U1 snRNP failed to be enriched in U1-A-immunoprecipitated RNA as demonstrated by RIP assay (upper panel). Electrophoresis analysis result of the qPCR products of immunoprecipitated RNA is shown (lower panel). J Blockage of U1 snRNA with U1 AMO abrogated the cis-regulatory effect of circME1 at mRNA level of ME1 as demonstrated by qPCR assay.