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The timing of splicing varies significantly between introns, transcripts, genes and species. Although quick co-transcriptional intron removal has been demonstrated for many mammalian genes, most splicing events do not occur immediately after intron synthesis. In this study, we utilized the highly expressed Tg gene, which forms exceptionally long transcription loops, providing a convenient model for studying splicing dynamics using advanced light microscopy. Using single-cell oligopainting, we observed a splicing delay occurring several tens of kilobases downstream of a transcribed intron, a finding supported by standard cell population analyses. We speculate that this phenomenon is due to the abnormally high transcriptional rate of the Tg gene, which might lead to a localized deficiency in splicing factors and, consequently, delayed spliceosome assembly on thousands of nascent transcripts decorating the gene. Additionally, we found that, in contrast to what is seen for short introns (&amp;lt;10\u2005kb), the long Tg intron (&amp;gt;50\u2005kb) is spliced promptly, providing further support for the idea that intron length might modulate splicing speed.<\/jats:p>","DOI":"10.1242\/jcs.263872","type":"journal-article","created":{"date-parts":[[2025,3,19]],"date-time":"2025-03-19T10:45:03Z","timestamp":1742381103000},"source":"Crossref","is-referenced-by-count":0,"title":["Co-transcriptional splicing is delayed in the highly expressed thyroglobulin gene"],"prefix":"10.1242","volume":"138","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1765-1251","authenticated-orcid":false,"given":"Simon","family":"Ullrich","sequence":"first","affiliation":[{"name":"Ludwig Maximilians University Munich 1 Faculty of Biology , , 82152 Planegg-Martinsried ,","place":["Germany"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Iliya","family":"Nadelson","sequence":"additional","affiliation":[{"name":"Ludwig Maximilians University Munich 1 Faculty of Biology , , 82152 Planegg-Martinsried ,","place":["Germany"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Stefan","family":"Krebs","sequence":"additional","affiliation":[{"name":"Gene Center, Ludwig Maximilians University Munich 2 Laboratory for Functional Genome Analysis (LAFUGA) , , 81377 Munich ,","place":["Germany"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Helmut","family":"Blum","sequence":"additional","affiliation":[{"name":"Gene Center, Ludwig Maximilians University Munich 2 Laboratory for Functional Genome Analysis (LAFUGA) , , 81377 Munich ,","place":["Germany"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Heinrich","family":"Leonhardt","sequence":"additional","affiliation":[{"name":"Ludwig Maximilians University Munich 1 Faculty of Biology , , 82152 Planegg-Martinsried ,","place":["Germany"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6813-7279","authenticated-orcid":false,"given":"Irina","family":"Solovei","sequence":"additional","affiliation":[{"name":"Ludwig Maximilians University Munich 1 Faculty of Biology , , 82152 Planegg-Martinsried ,","place":["Germany"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"237","published-online":{"date-parts":[[2025,3,19]]},"reference":[{"key":"2025032605465860500_JCS263872C2","doi-asserted-by":"publisher","first-page":"e1401","DOI":"10.1002\/wrna.1401","article-title":"Perfect timing: splicing and transcription rates in living cells","volume":"8","author":"Alpert","year":"2017","journal-title":"WIREs RNA"},{"key":"2025032605465860500_JCS263872C3","doi-asserted-by":"publisher","first-page":"1774","DOI":"10.1101\/gr.177790.114","article-title":"Widespread intron retention in mammals functionally tunes transcriptomes","volume":"24","author":"Braunschweig","year":"2014","journal-title":"Genome Res."},{"key":"2025032605465860500_JCS263872C4","doi-asserted-by":"publisher","first-page":"e1000573","DOI":"10.1371\/journal.pbio.1000573","article-title":"The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing","volume":"9","author":"Brody","year":"2011","journal-title":"PLoS Biol."},{"key":"2025032605465860500_JCS263872C5","first-page":"9","article-title":"Counting on co-transcriptional splicing","volume":"5","author":"Brugiolo","year":"2013","journal-title":"F1000Prime Rep."},{"key":"2025032605465860500_JCS263872C6","doi-asserted-by":"publisher","first-page":"395","DOI":"10.1016\/B978-0-12-420138-5.00021-5","article-title":"Experimenters\u2019 guide to colocalization studies: finding a way through indicators and quantifiers, in practice","volume":"123","author":"Cordelieres","year":"2014","journal-title":"Methods Cell Biol."},{"key":"2025032605465860500_JCS263872C8","doi-asserted-by":"publisher","first-page":"1845","DOI":"10.1126\/science.1162228","article-title":"Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters","volume":"322","author":"Core","year":"2008","journal-title":"Science"},{"key":"2025032605465860500_JCS263872C9","doi-asserted-by":"publisher","first-page":"RP91357","DOI":"10.7554\/eLife.91357.3","article-title":"Post-transcriptional splicing can occur in a slow-moving zone around the gene","volume":"12","author":"Cot\u00e9","year":"2024","journal-title":"eLife"},{"key":"2025032605465860500_JCS263872C10","doi-asserted-by":"publisher","first-page":"205","DOI":"10.1007\/978-1-59745-406-3_15","article-title":"Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes","volume":"463","author":"Cremer","year":"2008","journal-title":"Methods Mol. 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