Abstract
Widespread cotranscriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. Here, we adapted native elongating transcript sequencing technology (NET-seq) to measure cotranscriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. Our results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. We found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nt of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. We studied the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. We further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes, Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, our data unravel novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.
Highlights
We used native elongating transcript sequencing technology (NET-seq) to map polymerase II (Pol II) with the carboxy-terminal domain (CTD) phosphorylated on either S5 or S2 positions over the bodies of genes that become transcriptionally active in Drosophila embryos during the initial stages of development
Early Drosophila embryos contain abundant mRNAs that are transcribed during oogenesis and loaded into the egg
In our analysis of early embryos, these maternal transcripts were readily detected by RNA-seq, but not by dNET-seq
Summary
It is largely accepted that splicing can happen, and often does happen, while transcription is still in progress (Beyer and Osheim 1988; Carrillo Oesterreich et al 2010; Ameur et al 2011; Khodor et al 2011; Windhager et al 2012; Brugiolo et al 2013; Nojima et al 2015; Alpert et al 2017). Previous attempts to determine the duration of splicing in vivo had returned estimates ranging from a few seconds to several minutes (Alpert et al 2017). The lowest of these estimates, such as the few-second estimate reported in Martin et al (2013), are consistent with
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