The temporal dynamics of splicing can vary significantly − even among introns in the same nascent mRNA. Some introns are quickly removed co-transcriptionally whereas others might be delayed or excised non-sequentially. Determining factors that influence the timing of splicing is challenging because splicing events are difficult to observe in a live-cell context. In this study (Ullrich et al., 2025), Irina Solovei and colleagues investigate splicing dynamics of the highly expressed thyroglobulin gene (Tg) in mouse thyroid sections. In thyrocytes, Tg transcription elicits massive accumulation of RNA polymerase and splicing machinery along the gene, forming micron-scale transcription loops that can be resolved by conventional light microscopy. Using RNA fluorescence in situ hybridisation to label individual introns at Tg transcription loops, the authors find unspliced introns in a high proportion of nascent Tg mRNA. By observing colocalisation of simultaneously labelled sequential introns, they confirm splicing delays in Tg mRNA but not in transcripts of a similarly sized but moderately expressed gene. Interestingly, although short Tg introns (<10 kb) exhibit varying degrees of colocalisation with introns up to 50–60 kb downstream, the longest Tg intron (>50 kb) shows minimal colocalisation with subsequent introns, indicating it is spliced more efficiently than its short counterparts immediately upstream or downstream. Based on these findings, the authors propose that intron length influences splicing speed and that extensive transcription impacts splicing dynamics, with delayed intron removal potentially resulting from the local depletion of splicing machinery.
