Translational Profiling of Clock Cells Reveals Circadianly Synchronized Protein Synthesis

<div><p>Abstract</p><p>Genome-wide studies of circadian transcription or mRNA translation have been hindered by the presence of heterogeneous cell populations in complex tissues such as the nervous system. We describe here the use of a <i>Drosophila</i> cell-specific translational profiling approach to document the rhythmic “translatome” of neural clock cells for the first time in any organism. Unexpectedly, translation of most clock-regulated transcripts—as assayed by mRNA ribosome association—occurs at one of two predominant circadian phases, midday or mid-night, times of behavioral quiescence; mRNAs encoding similar cellular functions are translated at the same time of day. Our analysis also indicates that fundamental cellular processes—metabolism, energy production, redox state (e.g., the thioredoxin system), cell growth, signaling and others—are rhythmically modulated within clock cells via synchronized protein synthesis. Our approach is validated by the identification of mRNAs known to exhibit circadian changes in abundance and the discovery of hundreds of novel mRNAs that show translational rhythms. This includes <i>Tdc</i>2, encoding a neurotransmitter synthetic enzyme, which we demonstrate is required within clock neurons for normal circadian locomotor activity.</p></div

Mutation of the <i>Tdc2</i> gene results in decreased activity and circadian arrhythmicity for adult locomotor activity.

<p>(A) Quantification of average activity level, average rhythmicity index (RI), and percent of rhythmic flies in wild-type and <i>Tdc2^RO54</i> populations. <i>n</i> = 25 for control; <i>n</i> = 29 for <i>Tdc2^RO54</i>. Error bars represent SEM. *<i>p</i><0.0001. (B) Representative actograms, mean activity, and correlograms for control flies and the <i>Tdc2^RO54</i> mutant.</p

Biological processes represented by the rhythmically translated mRNAs.

<p>(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.</p

Expression of EGFP-L10a and assays of function in clock cells.

<p>(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 <i>elav-Gal4</i> pan-neuronal driver. (F) Expression of EGFP-L10a in all clock cells driven by <i>tim-Gal4</i>. (G) Restricted expression of EGFP-L10a to clock neuron but not glia using a combination of <i>tim-Gal4</i> and <i>repo-Gal80</i>. (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 <i>pdf-Gal4</i>) or all clock cells (using <i>tim-Gal4</i>). 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, <i>w¹¹¹⁸; act5C-Gal4/tub-Gal80^ts; UAS-EGFP-L10a/+</i>. IRP overexpression, <i>w¹¹¹⁸; act5C-Gal4/tub-Gal80^ts; UAS-EGFP-L10a/UAS-IRP</i>. (J) Circadian changes in the translation of period (<i>per</i>) and timeless (<i>tim</i>) mRNAs. Genotype of the flies assayed, <i>elav-Gal4; UAS-EGFP-L10a/+</i>. Error bar represents standard error of the mean (SEM). *<i>p</i><0.01; **<i>p</i><0.001 (Student's <i>t</i> test).</p

TDC2 protein shows circadian changes in the PDF-positive large ventral lateral neurons (l-LNvs) and dorsal lateral neurons (LNds).

<p>(A–B) Translational profile of <i>Tdc2</i> 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. <i>n</i>≥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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001703#s5" target="_blank">Materials and Methods</a> 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. *<i>p</i><0.01; **<i>p</i><1.5E-05 based on paired Student's <i>t</i> test.</p

Knockdown of <i>Tdc2</i> in clock neurons results in circadian behavioral arrhythmicity.

<p>(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. *<i>p</i><1.4E-30 for comparison with the control groups based on Student's <i>t</i> test.</p

TRAP identifies two major phases of rhythmic translation.

<p>(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). <i>n</i> = 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).</p