Comparative transcriptome-translatome analyses in the Drosophila head.

(A) Schematics. Fly head lysate is digested with RNase I for Ribo-seq, while not for RNA-seq. Resultant short fragments or the whole mRNA are reverse-transcribed and sequenced. (B) Meta-genome ribosome distribution (estimated P-sites of the 21-nt fragments), relative to the annotated start and stop codons. RPM: reads per million. (C) Scatter plots of mRNA reads (x-axis, TPM: transcripts per million) and ribosome footprints on CDS (y-axis, TPM). Several neuron-related genes are highlighted with colors and arrows. The squared Pearson’s correlation coefficient (R2) is indicated. (D-F) Ribosome footprints (D), mRNA level (E), and translational efficiency (F) of Shaker-RB (Sh) and Trehalase-RA (Treh). TE: translational efficiency. TE is calculated as ribosome footprints on CDS (TPM) divided by the mRNA level (TPM). (G) Histogram of TE. The bin size is 0.2 in the unit of log 2. Total 9,611 genes with at least one read in both Ribo-seq and RNA-seq are plotted. (H) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis, visualized by iPAGE (Goodarzi et al., 2009), based on TE. The 9,611 genes are ranked and binned according to TE (left to right: low to high), and over- and under-representation is tested. The presented KEGG pathways show P values smaller than 0.0005. (I) TE of transcripts in the denoted gene ontology terms. Bars represent the median. ns: P > 0.05; ***: P < 0.001; in the Dunn’s multiple comparisons test, compared to the “all” group.

Cell-type specific Ribo-seq and RNA-seq reveal differential translational regulations.

(A) Schematics. FLAG-tagged ribosome protein L3 (RpL3::FLAG) is expressed in neurons (nSyb-GAL4) or in glial cells (repo-GAL4). RNA-seq and Ribo-seq are performed following immunoprecipitation. Whole brain images of the exogenously expressed RpL3::FLAG are shown. Scale bars: 50 µm. (B) The MA-plot of ribosome footprints on CDS among neurons and glia. Each gene is plotted according to the fold change (x-axis) and the average (y-axis) in the unit of log2. Several marker genes are highlighted with green (neuron) or blue (glia). (C) TE of genes in the denoted KEGG pathways in the whole head (black), neurons (green), or in glia (blue). Genes with TPM > 1 in the RNA-seq dataset are plotted. Bars represent the median. *: P < 0.05, **: P < 0.01, ***: P < 0.001, the Dunn’s multiple comparisons test. (D) KEGG pathway enrichment analysis based on the ratio of TE in neurons to in glia. All genes with at least one read in both cell types (total 9,732 genes) are ranked and binned according to the neuron-to-glia ratio (left to right: high to low), and over- and under-representation is tested. The presented KEGG pathways show P values smaller than 0.0005. (E) Scatter plot of TE in neurons (x-axis) and in glia (y-axis). The squared Pearson’s correlation coefficient (R2) is indicated. (F) TE in glia plotted according to the ratio of mRNA expression in neurons compared to glia. ***: P < 0.001, Kruskal-Wallis test. All the 7,933 genes showing TPM > 1 in RNA-seq are analyzed. (G) TE of transcripts, showing at least one read, in the indicated gene ontology (GO) terms. Bars represent the median. **: P < 0.01, ***: P < 0.001, Dunn’s multiple comparisons test. (H) Read counts of genes (TPM) in the indicated GO terms in RNA-seq (yellow) and in Ribo-seq (pink). The grey, green and blue dots indicate the read counts in the whole head, neurons and glial cells, respectively. ns: P > 0.05, **: P < 0.01, ***: P < 0.001, Dunn’s multiple comparisons test.

Ribosome stalling on the 5′ leaders of DTTs in glia.

(A) Ribosome distribution (estimated P-sites) on the 161 DTTs around the start codons (solid lines; start +/- 50 nt). These DTTs are defined as transcripts showing more than 10 times higher TE in neurons compared to glia. The dotted lines in the bottom graph indicate the genome-wide distribution. All the transcripts showing TPM > 1 in RNA-seq both in neurons and glia are considered (7,933 genes in total), and the height is normalized by the total reads on this region. (B) Ratio of ribosome density on 5′ leader (TPM) to CDS (TPM) of the 161 DTTs or of all transcripts in neurons (green) or in glia (blue). The bars represent the median. ***: P < 0.001, Mann-Whitney test of ranks. (C) Distribution of ribosome footprints on the representative neuronal transcripts. Ribosome footprints (RPM) normalized by the mRNA level (TPM) are shown. Note that Syn-RD harbors a stop codon in the CDS but a fraction of ribosomes skip it, generating two annotated ORFs (CDS1 and CDS2) (Klagges et al., 1996). (D) Ratio of ribosome density on 5′ leader to CDS (mean ± standard error of mean of the biological replicates). (E) Ratio of ribosome density on 5′ leader to CDS on transcripts in the indicated GO terms in glia. *: P < 0.05, ***: P < 0.001, Dunn’s multiple comparisons test compared to the “all” group.

Footprint accumulation on upstream AUG in glia.

(A) Meta-genome ribosome distribution (estimated P-sites of the 32-nt fragments) around the upstream AUG codons in glia. (B) Meta-genome ribosome distribution (estimated P-sites of the 32-nt fragments) around the annotated start codons in glia. (C) Footprint accumulation on 5′ leader in glia, defined as the number of ribosome footprints (estimated P-sites) on each codon normalized by the average on the surrounding (−50 to +50) regions. (D) Footprint accumulation inside the annotated CDS in glia. Annotated in-frame codons except the start and the stop codons are considered. AU: arbitrary unit.

The transgenic Rh1-Venus reporter reveals differential translation in neuronal and glial cells.

(A) Reads on CDS of Rh1-RA in Ribo-seq. (B) Ribosome distribution (estimated P-sites) on Rh1-RA in neurons (green) and in glia (blue), with 0 on the x-axis indicating the start codon of the CDS. 6-base upstream ORFs, consisting of consecutive start (or the near-cognate) and stop codons, are highlighted. Note that footprints are normalized by the mRNA level (TPM). (C) Ratio of ribosome density on 5′ leader (TPM) to CDS (TPM) in neurons (green) or in glia (blue). The bars and the dots represent the median and individual data points, respectively. (D) Schematics of the control (UASz-GFP) or the Rh1 (UASz-Rh1-Venus) reporter. For the Rh1 reporter, 5′ leader and 3′ UTR sequences of Rh1-RA are fused to CDS of the Venus fluorescent protein. For the control reporter, synthetic 5′ leader sequences (syn21) and viral p10 terminator are fused to GFP (DeLuca and Spradling, 2018). Note that both reporters contain the same promoter (UASz) (DeLuca and Spradling, 2018) and are inserted onto the identical genomic locus (attP40). (E) Expression of the Rh1- or the control reporters driven by Tubulin-GAL4. Sliced confocal images of the cortical regions next to the antennal lobe are shown. Green: EGFP or Venus fluorescent signal. Red: Immunohistochemical signal of repo protein as a glial marker. Grey: EGFP or Venus mRNA. Orange arrowheads indicate glial cells marked by the repo expression. Scale bars: 5 µm. (F) Quantification of the green fluorescent intensity in glial nuclei, nomalized by the fluorescence in neurons. Glial intensity was measured as mean intensity in the repo-positive pixels, and was normalized by the mean intensity in the repo-negative pixels. **: P < 0.01, Mann-Whitney test of ranks. (G) Schematics of the mutated Rh1 reporter (m-Rh1). The minimal uORF is replaced with CCCAAA. (H) The expression of the Rh1- or m-Rh1- reporters, driven by the nSyb- or the repo- GAL4. Sliced confocal images of the cortical regions next to the antennal lobe are shown. Green: Venus fluorescent signal. Red: Venus mRNA signal. The total protein signal was normalized by the total mRNA signal for each brain. N = 8 (nSub > Rh1), 8 (nSyb > m-Rh1), 16 (repo > Rh1), 13 (repo > m-Rh1). ns: P > 0.05, *: P < 0.05, Mann-Whitney test of ranks.