MicroRNAs shape circadian hepatic gene expression on a transcriptome-wide scale
- University of Lausanne, Switzerland
- Vital-IT, Swiss Institute of Bioinformatics, Switzerland
Figures
Figure 1 with 2 supplements
Analysis of hepatic Dicer knockout using miR-122 as a diagnostic marker.
(A) Schematic of the Dicer knockout protocol used throughout the study. Conditional knockout and control littermates (heterozygotes and wild-type) carrying the AlbCre–ERT2 allele that ensures …
Figure 1—figure supplement 1
Kinetics of Dicer knockout in liver.
(A) PCR analysis of recombination kinetics at the Dicer locus. Male and female mice homozygous for the Dicerflox allele (also carrying AlbCre-ERT2) were injected with tamoxifen on five consecutive …
Figure 1—figure supplement 2
Global analysis of miRNA depletion in Dicer knockout livers.
(A) miRNA microarray analysis of wild-type vs heterozygote knockout liver RNA shows that a single functional copy of Dicer is sufficient to process miRNAs to wild-type levels. No significantly …
Figure 2 with 3 supplements
Genome-wide quantification of pre-mRNA and mRNA abundance by RNA-seq.
(A) Summary of RNA-seq results from Dicer KO (left) and control (right). Percentages are relative to total reads across all time points. (B) Time-resolved analysis of intron- (yellow) and …
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Figure 2—source data 1
RNA-seq read statistics.
- https://doi.org/10.7554/eLife.02510.007
Figure 2—figure supplement 1
Analysis of RNA-seq data by RNA classes.
Distribution of RNA-seq reads in Dicer knockout (red) and control mice (blue) to different RNA classes. Percentages represent mean ± standard deviation across the 12 samples per genotype. …
Figure 2—figure supplement 2
Reads mapping to the Dicer locus confirm high knockout efficiency.
(A) Distribution of RNA-seq reads in knockouts (red) and controls (blue) across the portion of the Dicer locus that carries the loxP-flanked exon 24, which contains part of the catalytic domain of Di…
Figure 2—figure supplement 3
Several genes show extreme transcriptional changes in Dicer knockouts.
(A) A number of genes showed strikingly different transcription in Dicer knockouts, including several imprinted loci such as Igf2 or Cdkn1c. (B) Strong up-regulation was observed for a group of …
Figure 3 with 1 supplement
The hepatic core clock is remarkably resilient to miRNA loss.
(A) mRNA/pre-mRNA ratio analysis of core clock transcripts indicates that Per1, Per2, and Per3 have a significantly increased ratio in Dicer knockouts. (B) Time-resolved RNA-seq data for selected …
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Figure 3—source data 1
Core clock genes.
- https://doi.org/10.7554/eLife.02510.012
Figure 3—figure supplement 1
Core clock gene expression in Dicer knockouts.
Apart from Per1, Per2, and Cry2, core clock gene expression shows very little post-transcriptional changes upon miRNA loss, indicating that these transcripts are not miRNA targets in the liver. Note …
Figure 4 with 5 supplements
miRNAs may drive the rhythmic accumulation of a small set of transcripts.
(A) Venn diagram summarising the extent of rhythmicity detected on all levels, that is mRNA and pre-mRNA in Dicer KO and control. A 1.5-fold threshold on peak-to-trough ratio amplitudes estimated …
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Figure 4—source data 1
Rhythm parameters transcriptome-wide.
- https://doi.org/10.7554/eLife.02510.015
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Figure 4—source data 2
GO term analysis Dicer rhythmic transcripts.
- https://doi.org/10.7554/eLife.02510.016
Figure 4—figure supplement 1
Transcriptome-wide rhythmicity detection in Dicer knockouts and controls.
(A) Venn diagram summarising the extent of rhythmicity detected at all levels, that is mRNA and pre-mRNA in Dicer KO and control. A 1.5-fold threshold on peak-to-trough ratio amplitude estimated …
Figure 4—figure supplement 2
Differences in detected rhythmic transcripts between Dicer knockout and control are not caused by too low stringency rhythm detection.
(A–H) Venn diagrams summarising the extent of rhythmicity detected on all levels, that is mRNA and pre-mRNA in Dicer KO and control, using different parameters for rhythm detection. Variables were: …
Figure 4—figure supplement 3
The expression of genes linked to the cell cycle/DNA replication becomes rhythmic in Dicer knockout livers.
Likely due to the higher cellular turnover and regeneration rate in Dicer knockout livers, the expression of many genes involved in the regulation of the cell cycle is increased and their rhythms …
Figure 4—figure supplement 4
miRNAs as potential drivers of rhythmic mRNA accumulation.
Shown are further examples (next to those in Figure 4) of transcripts that are transcriptionally non-rhythmic, but show mRNA rhythms that are Dicer-dependent, indicating that miRNAs could be …
Figure 4—figure supplement 5
Quantitative real-time PCR analysis of several transcripts shown in main Figures 4 and 5.
Shown are mean values of two time series (filled circle and triangle), and the individual data points (open circle and triangle) plotted together in the same graph. (A) Stx2 and Uba6 are candidates …
Figure 5 with 2 supplements
miRNAs adjust the mRNA phases and amplitudes of rhythmically transcribed genes.
(A) Schematic representation of how mRNA stability affects circadian transcript accumulation. mRNAs with short half-lives (blue) will thus peak relatively early after the transcriptional peak …
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Figure 5—source data 1
Rhythmic transcripts used in Figure 5.
- https://doi.org/10.7554/eLife.02510.023
Figure 5—figure supplement 1
Analysis of phase and amplitude changes occurring upon Dicer knockout.
Similar to Figure 5C,D (which shows the differences in mRNA-to-pre-mRNA phase delay and mRNA/pre-mRNA amplitude change, respectively, in Dicer knockout vs control), these graphs analyse mRNAs only (A…
Figure 5—figure supplement 2
Permutation of the RNA-seq data set confirmed that the effects observed on the phases and amplitudes (main Figure 5C,D) are highly significant and specific to the Dicer knockout.
Two different permutation analyses were performed. First (panels A and B), independently for each gene the 12 timepoints were randomly permutated 10,130 times. For each individual permutation, the …
Figure 6 with 1 supplement
Regulation of circadian output pathways by specific miRNAs.
(A) GO term analysis identifies specific pathways enriched in the group of circadian transcripts that are likely miRNA-regulated (higher mRNA/pre-mRNA ratio in Dicer knockouts) but not so in the …
Figure 6—figure supplement 1
Serum analyses in Dicer knockouts indicate metabolic defects.
Analysis of (A) total cholesterol, (B) HDL cholesterol, (C) LDL cholesterol, (D) triglycerides, (E) glucose, and (F) serum lipase activity in the serum of 12 control and 13 Dicer knockout animals. …
Figure 7
Model, summary, and speculations.
In the liver, we propose that miRNAs play three distinct roles in the regulation of rhythmic gene expression. First, around 30% of rhythmically transcribed genes appear to be also regulated by …
