The developmental transcriptome of Drosophila melanogaster

Abstract

Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.

Figure 1. Discovery of new RNAs in the bithorax complex

Genomic organization and experimental evidence for new transcripts located between the HOX genes, abd-A and Abd-B based on short poly(A)+ RNA and total RNA-seq expression profiles. The numbers to the left of each track indicate the maximal number of reads for that sample. Three manually curated junction-based transcript models are shown, the green transcript model was fully validated by a cDNA, MIP06894.

Figure 2. Discovery of small non-coding RNAs

a. Poly(A)+ (yellow) and total RNA (blue) data from 10-12 h embryos are shown for the gp210 gene which hosts a representative new snoRNA. The maximal number of reads in the poly(A)+ and total RNA-Seq data are shown on the left and right of the track, respectively. b. The predicted RNA secondary structure of snoRNA_gp210 is characteristic of a H/ACA-box snoRNA. Nucleotides that are 100% conserved in sequence or base-pairing are indicated in green and blue, respectively. c. Embryonic expression of the new small RNAs. The scale bar indicates FPKM Z-scores.

Figure 3. Dynamics of Gene Expression

a. Transcripts expressed (FPKM>1) in the short poly(A)+ RNA-Seq data, FB5.12 (blue), modENCODE (purple). The bar graphs indicate the number of transcripts expressed in each sample (Supplementary Table 1), and the lines, the cumulative number of expressed transcripts. The lighter blue and purple lines indicate the cumulative number of transcripts expressed in the embryonic Total RNA-Seq samples. The horizontal dotted lines indicate the number of expressed previously annotated transcripts. b. Scatter plot of sex-biased gene expression. light red: female-biased annotated (n=960), dark red: female-biased NTRs (n=12), light blue: male-biased annotated (n=2,401), dark blue: male-biased NTRs (n=431), light grey: unbiased annotated (n=8,217), black: unbiased NTRs (n=136). c. Genome Coverage. For each developmental sample, the short poly(A)+ reads were used to estimate the percent of the genome covered using a cutoff of two reads. The mature and primary transcripts were inferred for the previously FB5.12 (dotted lines) and modENCODE (solid lines) gene models.

Figure 4. Developmentally regulated splicing events

a. Conservation of internal constitutive and cassette exons >50 nt that were annotated or new discoveries. (Annotated Constitutive, n=26,127; Annotated Cassette, n=438; modENCODE Cassette n=173; modENCODE Constitutive n=306; 5.12 Constitutive to modENCODE Cassette n=304). b. Clusters of regulated cassette exon events during development. The scale bar indicates Z-scores of Ψ. c. Regulated alternative splicing in CadN during embryogenesis. The maximal number of reads in the poly(A)+ RNA-Seq data are indicated for each track

Figure 5. Discovery of RNA editing events

a. Rows represent an edited sites. Rank ordered expression levels (number of reads) are shown in green and the rank ordered editing ratios are shown in red. Pictogram representations of editing motifs A, B, and C are shown. b. RNA editing of qvr. Male and female expression and conservation tracks are shown above RNA-Seq reads from adult females that align to the edited positions (orange). Conceptual translation of the unedited and fully transcripts result in four amino acid changes (red) at the C-terminus of QVR.