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. 2023 Jul 27;14(7):475. doi: 10.1038/s41419-023-06008-3

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© The Author(s) 2023

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 FISH images show THOC3 and PFKFB4 mRNA localization in H520/H1703 cells (40× magnification). Scale bars = 100 μm. B FISH images show localization of THOC3 and PFKFB4 mRNA in shNC/shTHOC3 H1703 cells (40× magnification). Scale bars = 100 μm. C qRT-PCR analysis shows PFKFB4 mRNA expression in the nucleus and cytoplasm of THOC3 knockdown H520/H1703 cells compared to shNC-infected cells. D Enrichment of PFKFB4 mRNA in THOC3/YBX1 immunoprecipitates relative to IgG in H1703 shown using qRT-PCR. E RNA pull-down analysis followed by WB shows the binding between the biotin-labeled sense strand of PFKFB4 3′UTR and THOC3/YBX1 in H1703 cells, with no binding detected with the anti-sense strand. F Detection of PFKFB4 mRNA at indicated time points after actinomycin D treatment in H1703 cells transfected with si-NC, YBX1-si1/2, or THOC3-si1/2. G RNA pull-down analysis followed by WB shows the binding between the biotin-labeled PFKFB4 3′UTR and YBX1 in shNC/shTHOC3 infected H1703 cells. H qRT-PCR results show PFKFB4 mRNA enriched in YBX1 immunoprecipitates relative to the IgG in H1703 transfected by si-NC or si-NSUN2. I Biotin-labeled RNA pull-down and WB in H1703 cells show the binding between YBX1 and regions of PFKFB4 3′UTR with or without m5C modification. J Sanger sequencing results show parts of normal PFKFB4 sequences (left) and sequences after bisulfite conversion (right), where methylated C is framed in red. K TRiC folds THOC3 protein, which binds to PFKFB4 mRNA and brings it out of the nucleus. Upon arriving in the cytoplasm, THOC3 acts as a scaffold to recruit YBX1 to stabilize the transcript by recognizing m5C. THOC3 and YBX1 work together to promote PFKFB4 mRNA transcription. Overexpressed PFKFB4 increases glycolysis and promotes LUSC proliferation and migration. *P < 0.05; **P < 0.01; ***P < 0.001. Variables are presented as mean ± SD.