PMC

Examples of alternative splice isoforms regulated by per loss-of-function and time-of-day. The 5′ UTR and nearby coding exons are shown for lola (A) and PRL-1 (B). (Solid black bars) Exons; (blue lines) introns. (Red histograms, top) The depth of sequencing coverage for wild-type and per⁰ samples (lola) or ZT0 and ZT12 (PRL-1). Bar graphs or line graphs show the expression levels of individual exons (bottom).

Period alters RNA editing frequency in CG42613 and retinophilin. (A) Expression levels of CG43216-RC transcript are largely unchanged by period (B). The frequency of RNA editing in per⁰ is fivefold greater vs. wild-type (ANOVA, p < 5.5 × 10⁻¹¹, ANOVA, q < 5.0 × 10⁻⁹). Similar to CG42613, expression levels of retinophilin-RA transcript are not dramatically changed by period (C). The frequency of RNA editing at Chr3R:1062097 is dramatically decreased in per⁰ brains (ANOVA, p < 8.0 × 10⁻¹¹; ANOVA, q < 1.1 × 10⁻¹⁰) (D). (Error bars) Mean ± standard error of the mean.

Twenty-four-hour transcriptional rhythms. The fold-changes (FC) of median-normalized cycling transcripts (p < 0.05, Fisher's G-test) are plotted as a heatmap for LD (A) and DD (B) samples. Transcripts are ordered by phase, as measured by JTK_Cycle. The order of transcripts along the vertical axis is identical between wild-type and per⁰. Distribution of amplitudes (maximum expression divided by minimum expression) of cycling transcripts for LD (C) and DD (D) samples; (inset) individual amplitudes of transcripts encoding key circadian clock components. Molecular functions as annotated in FlyBase of cycling genes are displayed as a pie chart for LD (E) and DD (F) (p < 0.05 for both JTK_Cycle and Fisher's G-test).

The noncoding region of Nop60B (Uhg6) oscillates with peak expression at ZT6. (A) The Nop60B.a transcript includes six protein-coding exons and seven noncoding exons ([solid black bars] exons) separated by a large intron (thin blue line). Smaller introns separating exons 7–13 are excised from Nop60B.a transcripts and processed to form mature snoRNAs and snmRNAs. (Black boxes) Mature ncRNAs expressed at detectable levels; (gray boxes) undetectable ncRNAs. (Red histograms) The depth of sequencing coverage in this region at ZT0, ZT6 (peak), ZT12, and ZT18 (trough). (B) Expression levels of Nop60B's coding exons show weak or nonexistent circadian oscillations while the noncoding exons (C) and (the median-normalized) ncRNAs they encode (D) oscillate with peak expression at ZT6.

Multiple Uhg-family members oscillate with peak expression during the light phase. (A) Fold-changes of median-normalized expression levels of Uhg-family members are shown as a heatmap. (White and black bars) The LD regimen under which these samples were collected. The order of transcripts along the vertical axis is identical between wild-type and per⁰. Note that in per⁰ brains, peak Uhg expression is phase-delayed by ∼6 h. (B) The fold-changes of all snoRNAs and snmRNAs encoded by Uhg genes and expressed at a detectable level are median-normalized and displayed as a heatmap. (C) Expression levels of all cycling Uhg-family members in DD are damped relative to the light phase of LD. The light phase is defined as ZT0 and ZT6 for LD samples, and CT0, CT4, and CT8 for DD samples. (Error bars) Mean ± standard error of the mean.

Transcripts differentially regulated by period. Two-way ANOVA was used to identify transcripts differentially regulated by per⁰ in both non-poly(A)- (A) and poly(A)-amplified (B) data sets. Median-normalized expression levels have been sorted by fold change (FC) (average wild-type expression in RPKM divided by average per⁰ expression in RPKM) and are represented as a heatmap for each transcript (vertical axis) at each time point (horizontal axis) surveyed, with yellow indicating high levels of expression, and blue, low levels. White and black bars represent light and dark, respectively, in the LD environmental regimen under which these samples were collected. The order of transcripts along the vertical axis is identical in A and B. Tequila-RB (C) and Cyp4p1-RA (D) are examples of transcripts whose expression is significantly altered between wild-type (blue) and Period loss-of-function (red). (Error bars) Mean ± standard error of the mean.

Novel splicing events in the brain transcriptome. (A) The majority of gapped reads map to previously annotated splice junctions (left); however, the vast majority of unique splice junctions detected in the brain transcriptome have not been previously annotated (right). (Blue) Splice junctions previously detected by the modENCODE Consortium (). (Green) Novel splice junctions with (dark green) or without (light green) canonical 5′ and 3′ acceptor/donor splice sites. (B) The depth of coverage of unique splicing events is plotted as a histogram. Not surprisingly, the more abundant a splice junction, the more likely it has been previously annotated. (C) The molecular identity of novel splicing events within ion channel genes was manually curated, with relative abundance plotted as a pie chart.

Novel transcriptional start sites in clock genes. The 5′ UTR and nearby coding exons are shown for Clock (A) and timeless (D). (Solid black bars) Exons; (thin blue lines) introns. (Red histograms) The depth of sequencing coverage at ZT0 and ZT12; (dark blue brackets, top) the number of gapped reads spanning previously annotated splice junctions; (green brackets) the number of gapped reads spanning novel splice junctions. (Arrows, bottom) PCR primers used to assay for the presence of a given splicing event, with blue indicating previously annotated and green indicating novel. (E) Exon primer; (I) intron primer. The expression levels of individual exons and introns are shown for Clock (B) and timeless (E). (C,F) rtPCR was used to detect the presence of splicing events using indicated primer pairs that span a given junction in brains of independent biological replicates collected at ZT0 and ZT12.

RNA-seq accurately measures circadian transcriptional rhythms. (A) Locomotor activity rhythms were monitored using an automated infrared beam-break apparatus in wild-type and per⁰ flies entrained to a 12 h:12 h light:dark (LD) environment (N = 32 flies for each experiment). Plots are histograms of beam-breaks binned at a 20-min resolution. At the times indicated in red, brains were dissected and total RNA was purified for amplification and RNA-seq analysis. (B) Raw RNA-seq reads were aligned to the reference fly genome and transcriptome using the RUM read mapping algorithm, and expression levels (presented as reads per kilobase per million reads, RPKM) were calculated. Transcript expression levels exhibit a high degree of reproducibility between biological replicates with R-squared values on the order of 0.99. Expression profiles for Clock (C) and timeless (D) show consistent patterns of circadian oscillation in wild-type brains (solid lines) as measured by RNA-seq. Period-null mutation (dashed lines) disrupts the normal circadian rhythmicity of each of these transcripts. Clock and timeless oscillate in both LD (red) and DD (blue) with expected phases, and their amplitudes are damped in DD relative to LD, also as expected. The x-axis labels show the Zeitgeber time (ZT) for LD experiments and circadian time (CT) for wild-type Canton-S flies in DD.