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. 2019 Feb 19;9:2274. doi: 10.1038/s41598-018-36684-7

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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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Identification of mRNAs generated by trans-splicing of DUX4-s and dok7 mRNAs. (A) Diagram of primer set 3 (primers #310 and #309) designed to generate a RT-PCR product of ~1080 bp from trans-spliced mRNA containing two sequential dok7 coding sequences. (B) Diagram of primer set 4 (primers #270 and #309) designed to generate a RT-PCR product of ~730 bp from trans-spliced mRNA in which the DUX4-s coding sequence is upstream of (5′ to) the dok7 coding sequence. (C) Diagram of primer set 5 (primers #310 and #217) used to identify an alternative configuration of the trans-spliced mRNA in which dok7 is upstream of (5′to) DUX4-s. (D) All three sets of primers generated reverse transcriptase (RT) dependent bands of the expected sizes (arrows), indicating that all forms of the trans-spliced mRNAs were generated. Sequencing of the bands indicated by arrows confirmed that trans-splicing joined the donor 1 site in the V5 epitope sequence of one mRNA to the upstream acceptor 1 site in the 5′ region of the second mRNA, exactly as seen above for trans-splicing of two DUX4-s mRNAs, i.e., as in band “B” of Figs 5 and 6A,B.