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. 2018 Jan 4;9:51. doi: 10.1038/s41467-017-02495-z

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© The Author(s) 2017

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. 5 — RAN translation of GGGGCC repeats from C9ORF72 spliced intron. a Schematic of bicistronic splicing dual-luciferase reporters with C9ORF72 exons and intron. C9R-NLuc reporter is located in intron 1 and AUG-FLuc reporter is in exon 2 fused with the original C9ORF72 start codon. The positions of primers amplifying different RNA products were labeled. b Relative RAN translation products from Frame-GA and Frame-GP were compared to no-repeat control, after 24 h induction in HeLa Flp-In cells. NLuc signals were normalized to FLuc in each sample. c Immunoprecipitation using MYC antibody from cells expressing Neg-NLuc, C9R-NLuc of frame-GA, or GP in bicistronic splicing reporters, followed by immunoblotting with GA or GP antibody. d The relative translation level of C9R-NLuc in monocistronic, bicistronic, and bicistronic splicing reporters. The NLuc luciferase level in each cell line was normalized to the NLuc RNA level, which was quantified by qRT-PCR. e After 24 h induction of reporter genes, cells were fractionated to separate nucleus and cytoplasm. The levels of reporter spliced mRNA and unspliced pre-mRNA, Nluc (amplifying excised intron and unspliced pre-mRNA), GAPDH pre-mRNA, and the mitochondria RNA MTRNR1 were measured by qRT-PCR and normalized to GAPDH mRNA in each fraction. The ratio of cytosol/nuclear RNA showed the subcellular distribution of each RNA. The nuclear marker GAPDH pre-mRNA is depleted in cytosol fraction (left) and the cytosol marker MTRNR1 is highly enriched in cytosol fraction (middle). NLuc RNA shows more cytosolic distribution than pre-mRNAs (right). f The diagram of experiment design and TRAP methodology to isolate ribosome-associated RNAs. g Total, cytosol, and ribosome-associated RNAs were isolated for qRT-PCR. NLuc and reporter pre-mRNA were measured and normalized to GAPDH mRNA in each fraction. The ratios of ribosome-associated/cytosol RNA (left) and ribosome-associated/total RNA (right) show that the Nluc intron RNA is associated with ribosomes, but the pre-mRNA is not. Data are mean ± SEM from three biological replicates