Computational protocol: Translational Profiling of Clock Cells Reveals Circadianly Synchronized ProteinSynthesis

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Protocol publication

[…] Using the newly developed methods, we performed TRAP on head tissue lysates of tim-uas-Gal4; UAS-EGFP-L10a flies that were collected at 4-h intervals during the first two days of constant darkness (DD) following entrainment to LD 12∶12. Such flies express the EGFP-L10a fusion in all clock cells of the head, including the ∼150 pacemaker neurons, photoreceptors, and glia. RNA was extracted from affinity-purified samples and used to generate libraries representing all ribosome-associated transcripts (see ). TRAP libraries corresponding to six different times of the circadian cycle (CT0, 4, 8, 12, 16, and 20) were independently constructed for DD1 and DD2 (see details in ). Libraries were sequenced, using a multiplexing strategy, to produce single end, 100 base sequencing reads; these were mapped to the Drosophila reference genome and analyzed as described in .We employed two recently developed programs, JTK_CYCLE and ARSER ,, to compare their usefulness for detecting circadian rhythms in the ribosome association of mRNAs. Using criteria and statistical cutoffs described in the section, 1,195 and 263 translationally cycling mRNAs were detected by the ARSER and JTK_CYCLE programs, respectively. Interestingly, the majority of the cycling mRNAs (203 out of 263) detected by JTK_CYCLE were also detected by the ARSER program (), indicating consistency of the two analyses. shows robust cycling for eight mRNAs out of the 60 identified by JTK_CYCLE but not ARSER. Thus, JTK_CYCLE may identify cycling mRNAs not detected by ARSER. lists the 1,255 mRNAs that were identified as exhibiting significant translational cycling by either program (mRNAs identified by both programs are indicated in bold). The False Discovery Rate (FDR) calculated by the ARSER program at the relevant p value was 0.148, indicating that approximately 186 mRNAs are false positives. This FDR is quite low relative to other recent genome-wide studies of cycling mRNAs –. We did not compute an FDR for the JTK_CYCLE program, because 203/263 mRNAs identified by JTK_CYCLE are included in the ARSER dataset, and therefore the latter dataset represents a good approximation of FDR for our analyses. Based on the ARSER analysis, we estimate that approximately 1,069 of these mRNAs show circadian changes in translation in clock cells of the adult head, representing about 10% of all analyzed genes in the genome. This large number of cycling mRNAs is consistent with recent studies utilizing manual dissection approaches to perform cell-specific transcriptional profiling of the Drosophila PDF clock neurons ,. Cell-specific profiling methods may identify a larger number of cycling Drosophila mRNAs, relative to previous studies, due to a more homogeneous starting cell population (i.e., clock cells). […]

Pipeline specifications

Software tools JTK_CYCLE, ARSER
Application Transcription analysis
Organisms Drosophila melanogaster