show Abstracthide AbstractWidespread co-transcriptional splicing has been demonstrated from yeast to human. However, measuring the kinetics of splicing relative to transcription has been hampered by technical challenges. Here, we took advantage of native elongating transcript sequencing (NET-seq) to identify the position of RNA polymerase II (Pol II) when exons become ligated in the newly synthesized RNA. We analyzed Drosophila melanogaster embryos because the genes transcribed initially during development have few and short introns (like yeast genes), whereas genes transcribed later contain multiple long introns (more similar to human genes). Moreover, compared to human, the Drosophila genome is more compact and thus the coverage of NET-seq reads on intragenic regions is higher. We detected spliced NET-seq reads connected to Pol II molecules that were positioned just a few nucleotides downstream of the 3' splice site. Although the majority of splice junctions were covered by spliced reads, many introns remained unspliced, resulting in a complex range of heterogeneity in splicing dynamics. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. Both canonical and recursive splicing were associated with a higher Pol II density, suggesting a splicing-coupled mechanism that slows down transcription elongation. We further observed that transcription termination was very efficient for isolated genes but that the presence of an overlapping antisense gene was often associated with transcriptional read-through. Taken together, our data unravels novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo. Overall design: Native Elongating Transcript sequencing was performed in whole Drosophila embryos collected at one of two timepoints (2-3 hours post-fertilisation and 4-6 hours post-fertilisation).