Dynamic properties of transcription and co-transcriptional splicing during early stages of Drosophila development. Dynamic properties of transcription and co-transcriptional splicing during early stages of Drosophila development

Widespread 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).

共转录剪接（co-transcriptional splicing）的普遍性已在酵母至人类的多个物种中得到证实。然而，精准测定剪接相对于转录的动力学特征仍受限于诸多技术瓶颈。本研究借助天然延伸转录本测序（native elongating transcript sequencing, NET-seq）技术，定位新合成RNA中外显子发生连接时RNA聚合酶II（RNA polymerase II, Pol II）的具体位置。我们选取黑腹果蝇（Drosophila melanogaster）胚胎作为实验材料，原因在于发育早期转录的基因内含子数量少且长度较短（类似酵母基因），而发育后期转录的基因则包含多个长内含子（更接近人类基因）。此外，相较于人类基因组，果蝇基因组更为紧凑，因此NET-seq读段在基因区域的覆盖度更高。我们检测到与Pol II分子相连的剪接型NET-seq读段，这些Pol II分子恰好位于3'剪接位点（3' splice site）下游数个核苷酸的位置。尽管大多数剪接接头（splice junction）均有剪接型读段覆盖，但仍有大量内含子未发生剪接，这使得剪接动力学呈现出复杂的异质性特征。若内含子在Pol II抵达下游外显子末端前即完成剪接，则该内含子必然属于内含子定义型（intron-defined）。正如预期，我们发现剪接读段的占比与内含子长度存在相关性；但无论内含子长度如何，均能观察到内含子定义型剪接现象。经典剪接（canonical splicing）与递归剪接（recursive splicing）均与更高的Pol II密度相关，这提示存在一种耦合剪接的转录调控机制，可减缓转录延伸速度。我们还观察到，对于独立基因而言，转录终止效率极高；但当基因组存在重叠的反义基因时，往往会发生转录通读（transcriptional read-through）现象。综上，本研究的数据揭示了发育中的黑腹果蝇胚胎内Pol II转录与剪接的全新动力学特征。实验设计：本研究对两个时间点（受精后2-3小时、受精后4-6小时）收集的完整黑腹果蝇胚胎开展了天然延伸转录本测序。