Abstract:Widespread co-transcriptional 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 co-transcriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. Our results reveal the position of RNA polymerase II (Pol II) when both… Show more
“…We assume that introns are co-transcriptionally spliced and rapidly degraded. In support of this, NET-seq data suggests that >95% of splicing events are co-transcriptional in the Drosophila embryo [29] and modelling of metabolic labelling and sequencing data from Drosophila S2 cells revealed that the median half-life of introns is 2 minutes [30].…”
Section: Gaussian Process Regression Provides Estimates Of Transcript...mentioning
Regulation of mRNA degradation is critical for a diverse array of cellular processes and developmental cell fate decisions. Many methods for determining mRNA half-lives rely on transcriptional inhibition or metabolic labelling. Here, we use a non-invasive method for estimating half-lives for hundreds of mRNAs in the early Drosophila embryo. This approach uses the intronic and exonic reads from a total RNA-seq time series and Gaussian process regression to model the dynamics of premature and mature mRNAs. We show how regulation of mRNA stability is used to establish a range of mature mRNA dynamics during embryogenesis, despite shared transcription profiles. Using single-molecule imaging, we provide evidence that, for the mRNAs tested, there is a correlation between short half-life and mRNA association with P-bodies. Moreover, we detect an enrichment of mRNA 3′ ends in P-bodies in the early embryo, consistent with 5′ to 3′ degradation occurring in P-bodies for at least a subset of mRNAs. We discuss our findings in relation to recently published data suggesting that the primary function of P-bodies in other biological contexts is mRNA storage.
“…We assume that introns are co-transcriptionally spliced and rapidly degraded. In support of this, NET-seq data suggests that >95% of splicing events are co-transcriptional in the Drosophila embryo [29] and modelling of metabolic labelling and sequencing data from Drosophila S2 cells revealed that the median half-life of introns is 2 minutes [30].…”
Section: Gaussian Process Regression Provides Estimates Of Transcript...mentioning
Regulation of mRNA degradation is critical for a diverse array of cellular processes and developmental cell fate decisions. Many methods for determining mRNA half-lives rely on transcriptional inhibition or metabolic labelling. Here, we use a non-invasive method for estimating half-lives for hundreds of mRNAs in the early Drosophila embryo. This approach uses the intronic and exonic reads from a total RNA-seq time series and Gaussian process regression to model the dynamics of premature and mature mRNAs. We show how regulation of mRNA stability is used to establish a range of mature mRNA dynamics during embryogenesis, despite shared transcription profiles. Using single-molecule imaging, we provide evidence that, for the mRNAs tested, there is a correlation between short half-life and mRNA association with P-bodies. Moreover, we detect an enrichment of mRNA 3′ ends in P-bodies in the early embryo, consistent with 5′ to 3′ degradation occurring in P-bodies for at least a subset of mRNAs. We discuss our findings in relation to recently published data suggesting that the primary function of P-bodies in other biological contexts is mRNA storage.
“…We assume that introns are co-transcriptionally spliced and rapidly degraded. In support of this, NET-seq data suggests that >95% of splicing events are co-transcriptional in the Drosophila embryo [29] and modelling of metabolic labelling and sequencing data from Drosophila S2 cells revealed that the median half-life of introns is 2 min [30].…”
Section: Resultsmentioning
confidence: 98%
“…We assume that introns are spliced at the same rate for each mRNA, consistent with evidence from S2 cells that introns from the same mRNA tend to have similar splicing rates [30]. We normalised the read counts by the intron lengths so that the S parameter has comparable meaning for each mRNA, but allow it to differ from mRNA-to-mRNA to account for variation in splicing rates across genes [29] and also differences due to alternative splicing of different transcripts. For transcripts we used TPM units which normalises for transcript length.…”
Section: Resultsmentioning
confidence: 99%
“…In order to validate that the intronic data is representative of nascent zygotic gene transcription, NET-seq data from early embryos was used [29]. Both the NET-seq and intronic data were length normalised and a Spearman’s Rank correlation analysis was used to analyse the relationship.…”
Regulation of mRNA degradation is critical for a diverse array of cellular processes and developmental cell fate decisions. Although many methods for determining mRNA half-lives rely on transcriptional inhibition or metabolic labelling, these manipulations can influence half-lives or introduce errors in the measurements. Here we use a non-invasive method for estimating mRNA half-lives globally in the early Drosophila embryo using a total RNA-seq time series and Gaussian process regression to model the dynamics of premature and mature mRNAs. We show how regulation of mRNA stability is used to establish a range of mature mRNA dynamics during embryogenesis, despite shared transcription profiles. Our data suggest that mRNA half-life is not dependent on translation efficiency, but instead we provide evidence that short half-life mRNAs are more strongly associated with P-bodies. Moreover, we detect an enrichment of mRNA 3' ends in P-bodies in the early embryo, consistent with 5' to 3' degradation occurring in P-bodies for at least a subset of mRNAs. We discuss our findings in relation to recently published data suggesting that the primary function of P-bodies in other biological contexts is mRNA storage.
“…This allowed the decetion of splicing intermediates, such as exon 3ʹ-end and intron lariats, but prevented the analysis of promoter-proximal stalling. It thus seems that the two approaches can capture complementary information [ 40 ]. Figure 1.…”
DNA double-strand breaks are among the most toxic lesions that can occur in a genome and their faithful repair is thus of great importance. Recent findings have uncovered local transcription that initiates at the break and forms a non-coding transcript, called damage-induced long non-coding RNA (dilncRNA), which helps to coordinate the DNA transactions necessary for repair. We provide nascent RNA sequencing-based evidence that RNA polymerase II transcribes the dilncRNA in
Drosophila
and that this is more efficient for DNA breaks in an intron-containing gene, consistent with the higher damage-induced siRNA levels downstream of an intron. The spliceosome thus stimulates recruitment of RNA polymerase II to the break, rather than merely promoting the annealing of sense and antisense RNA to form the siRNA precursor. In contrast, RNA polymerase III nascent RNA libraries did not contain reads corresponding to the cleaved loci and selective inhibition of RNA polymerase III did not reduce the yield of damage-induced siRNAs. Finally, the damage-induced siRNA density was unchanged downstream of a T8 sequence, which terminates RNA polymerase III transcription. We thus found no evidence for a participation of RNA polymerase III in dilncRNA transcription in cultured
Drosophila
cells.
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