Nascent-Seq analysis of Drosophila cycling gene expression

Abstract

Rhythmic mRNA expression is a hallmark of circadian biology and has been described in numerous experimental systems including mammals. A small number of core clock gene mRNAs and a much larger number of output mRNAs are under circadian control. The rhythmic expression of core clock genes is regulated at the transcriptional level, and this regulation is important for the timekeeping mechanism. However, the relative contribution of transcriptional and post transcriptional regulation to global circadian mRNA oscillations is unknown. To address this issue in Drosophila, we isolated nascent RNA from adult fly heads collected at different time points and subjected it to high-throughput sequencing. mRNA was isolated and sequence din parallel. Some genes had cycling nascent RNAs with no cycling mRNA, caused,most likely, by light-mediated read-through transcription. Most genes with cycling mRNAs had significant nascent RNA cycling amplitudes, indicating a prominent role for circadian transcriptional regulation. However, a considerable fraction had higher mRNA amplitudes than nascent RNA amplitudes. The same comparison for core clock gene mRNAs gives rise to a qualitatively similar conclusion. The data therefore indicate a significant quantitative contribution of post transcriptional regulation to mRNA cycling.

Fig. 1.

Nascent-Seq identifies 136 transcriptional cyclers in Drosophila heads. (A) Overview of the workflow used to prepare and sequence rhythmic nascent RNA. (B) Cycling Nascent-Seq signal over the vri locus for one set of replicates. Signal within the intron is enriched. (C) The reads per base pair coverage (red) was used to quantify the average coverage within exons. The correlation of this measure between the replicate time points is high, with coefficients of R = 0.86 and R = 0.94 for ZT2 and ZT14, respectively. (D) Fourier analysis of Nascent-Seq datasets identifies 136 nascent cyclers. Shown are the Z-scores of the reads per base pair coverage plotted as a heatmap. (E) Two main peaks of expression are observed, at ZT4–ZT6 and ZT12–ZT18. (F) A few GO categories were enriched, including circadian rhythms and response to stimulus.

Fig. 2.

RNA-Seq identifies more than 237 mRNA cyclers in Drosophila heads. (A) Overview of the workflow used to prepare and sequence rhythmic mRNA. (B) Cycling mRNA signal over the vri locus for one set of replicates. Signal within the intron is depleted. (C) The reads per base pair coverage (blue) was used to quantify the average coverage within exons. The correlation of this measure between the replicate time points is good, with coefficients of R = 0.89 and R = 0.98 for ZT0 and ZT12, respectively. (D) Fourier analysis of RNA-Seq datasets identifies 237 mRNA cyclers. Shown are the Z-scores of the reads per base pair coverage plotted as a heatmap. (E) The phase distribution of cycling is more spread out than the Nascent-Seq cyclers and increases gradually, peaking at ZT22. (F) A few GO categories were enriched, including circadian rhythms and glutathione metabolism. (G) A significant overlap between the Nascent and mRNA cycling genes is observed. Forty-four genes have robust Nascent and mRNA cycling which is indicative of the classic circadian transcription model.

Fig. 3.

Four groups of cyclers are identified. (A) Four groups are identified based of cycling thresholds (Materials and Methods). Group I genes have robust nascent and mRNA cycling, whereas group II genes have robust nascent cycling but weak or flat temporal mRNA-expression profiles. Group III genes have robust cycling mRNA-expression profiles and weaker cycling nascent-expression profiles. Group IV genes have robust mRNA-expression profiles but very noisy or flat temporal nascent-expression profiles. Shown are the Z-scores of the reads per base pair coverage plotted as a heatmap. (B) Phase distribution of nascent RNA (red) and mRNA (blue) for the genes of groups I and II. Group II cycler mRNA phase distribution is not correlated with the nascent phase distribution, contrary to group I cyclers, which have very similar phase distributions. The phase was determined by assigning the time point with the maximum reads per base pair. (C) Group II genes have very weak mRNA expression compared with other group cyclers.

Fig. 4.

A subset of group II cyclers consists of read-through transcription at the beginning of the light phase. (A) In CG34321 and CG3454 the nascent cycling amplitude (red) is higher than the mRNA cycling amplitude (blue). Plotted are normalized reads per base pair averaged within exons. The data are double plotted. (B) A transcript appears to extend from the 3′ end of arr2 and transcribes across several downstream genes, which are located on both strands. No cycling signal is observed in the RNA-Seq data. Only the three day time points are shown for Nascent- and RNA- Seq profiles, in red and blue respectively. The expression profiles of the three night time points for the Nascent- and RNA-Seq datasets are indistinguishable from their ZT 10 and ZT 8 time points, respectively. (C) Zoomed-in view of ZT 2 Nascent-Seq read coverage (red) at the arr2 locus.

Fig. 5.

Posttranscriptional contribution to mRNA cycling. (A) RNA-Seq cycling amplitudes for genes in groups I, III, and IV are higher than Nascent-Seq amplitudes, indicating a posttranscriptional contribution to the gene-expression profile. The ratio of max/min reads per base pair coverage for each dataset was calculated from the averaged time points and was plotted. (B) Visualization of Nascent-Seq (red) and RNA-Seq (blue) data at the gol locus. gol is a group I cycler and has a higher mRNA cycling amplitude than nascent RNA cycling amplitude. (C) Visualization of Nascent-Seq (red) and RNA-Seq (blue) data at the CG4784 locus. CG4784 is a group IV cycler and has a higher mRNA cycling amplitude than nascent RNA cycling amplitude. (D) Visualization of Nascent-Seq (red) and RNA-Seq (blue) data at the CG10006 locus. CG10006 is a group IV cycler and has a higher mRNA cycling amplitude than nascent RNA cycling amplitude.

Fig. 6.

Validation of posttranscriptional candidates. Goliath, CG4784, and CG10006 cycling amplitudes are higher in the RNA-Seq data than in the Nascent-Seq data. All three candidates were validated by qRT-PCR using two independent samples each of Nascent and mRNA. An additional set of independent samples was assayed for gol and CG4784. Read per base pair quantitation of the sequencing data and of the qRT-PCR gene signal divided by RPL32 gene signal is plotted for each gene.

Fig. 7.

Some core clock genes also are under posttranscriptional control. By inspection, per and cry have higher mRNA cycling amplitudes relative to their respective nascent cycling amplitudes. vri also has a smaller posttranscriptional contribution to its mRNA cycling amplitude. See text for more detail.