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1.
Figure 5

Figure 5. Biological processes represented by the rhythmically translated mRNAs.. From: Translational Profiling of Clock Cells Reveals Circadianly Synchronized Protein Synthesis.

(A) Pie chart showing different represented processes. The number of mRNAs belonging to each category is shown next to each slice of the pie. (B) Translational profile of thioredoxin system mRNAs.

Yanmei Huang, et al. PLoS Biol. 2013 November;11(11):e1001703.
2.
Figure 2

Figure 2. Identification of mRNAs displaying a circadian translational rhythm in clock cells.. From: Translational Profiling of Clock Cells Reveals Circadianly Synchronized Protein Synthesis.

(A) Number of rhythmically translated genes identified by two different programs: JTK_CYCLE and ARSER. (B) Translational profile of known cycling genes. The y-axis represents normalized read counts. (C) Quantification of sequence reads aligned to the period (per) gene and a nearby nonrhythmic gene (CG2658) across the time-series.

Yanmei Huang, et al. PLoS Biol. 2013 November;11(11):e1001703.
3.
Figure 7

Figure 7. Mutation of the Tdc2 gene results in decreased activity and circadian arrhythmicity for adult locomotor activity.. From: Translational Profiling of Clock Cells Reveals Circadianly Synchronized Protein Synthesis.

(A) Quantification of average activity level, average rhythmicity index (RI), and percent of rhythmic flies in wild-type and Tdc2RO54 populations. n = 25 for control; n = 29 for Tdc2RO54. Error bars represent SEM. *p<0.0001. (B) Representative actograms, mean activity, and correlograms for control flies and the Tdc2RO54 mutant.

Yanmei Huang, et al. PLoS Biol. 2013 November;11(11):e1001703.
4.
Figure 8

Figure 8. Knockdown of Tdc2 in clock neurons results in circadian behavioral arrhythmicity.. From: Translational Profiling of Clock Cells Reveals Circadianly Synchronized Protein Synthesis.

(A–E) Representative actograms showing free-running locomotor activity of flies with a Tdc2 knockdown in PDF neurons (A) or all clock neurons (B), as well as relevant control files (C–E). (F) Quantification of the average rhythmicity index (RI) for various genotypes. Number of flies tested is indicated on the histograms. *p<1.4E-30 for comparison with the control groups based on Student's t test.

Yanmei Huang, et al. PLoS Biol. 2013 November;11(11):e1001703.
5.
Figure 4

Figure 4. Comparison of abundance and ribosome-association profiles for several mRNAs.. From: Translational Profiling of Clock Cells Reveals Circadianly Synchronized Protein Synthesis.

(A–C) Examples of mRNAs that show constant abundance but rhythms in ribosome association. (D) An example of an mRNA showing both abundance and ribosome association rhythms. RNA abundances were normalized to that of Rp49 for each time point. Abundance is expressed relative to that of the first time point (CT0), which was designated a value of 1. Negative and positive error bars show the range of possible relative values calculated based on the SEM of the Ct values obtained in the Q-PCR experiments. Each data point represents a sample size of 6 (3 biological replicates, each with 2 technical replicates).

Yanmei Huang, et al. PLoS Biol. 2013 November;11(11):e1001703.
6.
Figure 3

Figure 3. TRAP identifies two major phases of rhythmic translation.. From: Translational Profiling of Clock Cells Reveals Circadianly Synchronized Protein Synthesis.

(A, Upper) A heat map showing the relative level of translation during DD days 1–2 for each of the 1,255 genes. Genes are arranged vertically according to their phases. (A, Lower) Population plot of free-running activity (DD days 1–2) for the fly strain used to generate the translational profiles (vertical axis, activity level; horizontal axis, time of day). n = 17, error bars are SEM. (B) Phase distributions of ribosome association for all cycling RNAs. (C) Phase distributions of cycling RNAs relevant for several different cellular processes. Horizontal axes show phase; vertical axes indicate the number of RNAs. (D) Day or night distribution for major biological processes. (E) Translational profiles of mRNAs representing two functional groups: G protein–coupled receptors (upper panel) and glucose metabolic enzymes (lower panel).

Yanmei Huang, et al. PLoS Biol. 2013 November;11(11):e1001703.
7.
Figure 6

Figure 6. TDC2 protein shows circadian changes in the PDF-positive large ventral lateral neurons (l-LNvs) and dorsal lateral neurons (LNds).. From: Translational Profiling of Clock Cells Reveals Circadianly Synchronized Protein Synthesis.

(A–B) Translational profile of Tdc2 revealed by RNA sequencing (A) and Q-PCR (B). In the Q-PCR graph, the level of mRNA expression for the first time point (CT0) serves as a reference, and is thus designated a value of 1. RNA expression levels at other time points are plotted relative to the value at CT0. Negative and positive error bars show the range of possible relative values calculated based on the SEM of the Ct values obtained in the Q-PCR experiments. n≥4 for all time points. (C) Abundance of TDC2 protein in the l-LNvs and LNds at two different times of the circadian cycle, using immunohistochemical methods. (D) Sample images showing differential expression of TDC2 in l-LNvs (red channel) at ZT1 and ZT9. Quantification of average pixel intensities is described in the Materials and Methods section. For LNvs, 10 pairs of brain hemispheres were compared between ZT1 and ZT9. For LNds, nine pairs of brain hemispheres were compared between ZT1 and ZT9. *p<0.01; **p<1.5E-05 based on paired Student's t test.

Yanmei Huang, et al. PLoS Biol. 2013 November;11(11):e1001703.
8.
Figure 1

Figure 1. Expression of EGFP-L10a and assays of function in clock cells.. From: Translational Profiling of Clock Cells Reveals Circadianly Synchronized Protein Synthesis.

(A–C) Expression of EGFP-L10a in a large neurosecretory cell. Nu, nucleolus; N, Nucleus; C, Cytoplasm. Staining for a nuclear protein called LARK (red signal) is used to identify the nucleus. (D) Schematic representation of the structure of the nucleolus. FC, Fibrillar Center; DFC, Dense Fibrillar Components; GC, Granular Components. GC is the location of ribosome assembly. (E) Expression pattern of EGFP-L10a in the brain and ventral ganglion using the elav-Gal4 pan-neuronal driver. (F) Expression of EGFP-L10a in all clock cells driven by tim-Gal4. (G) Restricted expression of EGFP-L10a to clock neuron but not glia using a combination of tim-Gal4 and repo-Gal80. (H) Expression of EGFP-L10a in clock cells does not disrupt normal circadian behavior. Left panels shows representative free-running actograms of control flies and flies expressing EGFP-L10a in either PDF neurons (using pdf-Gal4) or all clock cells (using tim-Gal4). Right panels show the corresponding correlograms. (I) TRAP is capable of detecting changes in mRNA translation, as assayed by changes in the translational status of Ferritin 1 Heavy Chain Homolog (Fer1HCH) mRNA in response to overexpression of the Iron Regulatory Protein (IRP). Control, w1118; act5C-Gal4/tub-Gal80ts; UAS-EGFP-L10a/+. IRP overexpression, w1118; act5C-Gal4/tub-Gal80ts; UAS-EGFP-L10a/UAS-IRP. (J) Circadian changes in the translation of period (per) and timeless (tim) mRNAs. Genotype of the flies assayed, elav-Gal4; UAS-EGFP-L10a/+. Error bar represents standard error of the mean (SEM). *p<0.01; **p<0.001 (Student's t test).

Yanmei Huang, et al. PLoS Biol. 2013 November;11(11):e1001703.

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