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. 2020 Jan 14;9:e49658. doi: 10.7554/eLife.49658

Figure 6. Reduction in m6A RNA modification leads to disruption of the circadian clock.

(A) Genes with differential error rate sites have lower detectable RNA levels. Histogram showing the log2 fold change in protein coding gene expression based on counts from nanopore DRS reads in the vir-1 mutant compared to the VIR-complemented line. Blue, genes with differential error rate sites (n = 5083 genes); orange, genes without differential error rate sites (n = 14,675 genes). (B) The circadian period is lengthened in the vir-1 mutant. Mean delayed fluorescence measurements in arbitrary units: blue, Col-0 (n = 61 technical reps); green, the VIR-complemented line (VIRc; n = 29 technical reps); orange, vir-1 (n = 24 technical reps). Shaded areas show bootstrapped 95% confidence intervals for the mean. (C) Boxplot showing the period lengths calculated from delayed fluorescence measurements shown in (b): blue, Col-0; green, VIRc; orange, vir-1 (orange). (D) Poly(A) tail length is altered in the vir-1 mutant. Histogram showing the poly(A) tail length distribution of CAB1 (AT1G29930): blue, Col-0 (n = 40,841 reads); green, VIRc (n = 65,810 reads); orange, vir-1 (n = 68,068 reads). Arrows indicate phased peaks of poly(A) length at approximately 20, 40 and 60 nt. vir-1 distribution is significantly different from both Col-0 and VIRc, according to the Kolmogorov–Smirnov test (p < 1 × 10−16, p < 1 × 10−16 respectively).

Figure 6—source data 1. Differential gene expression results for vir-1 vs VIRc, using counts derived from nanopore DRS – Figure 6A.
Figure 6—source data 2. Delayed fluorescence and circadian period length for Col-0, vir-1 and VIRc – Figure 6B.
elife-49658-fig6-data2.xlsx (320.8KB, xlsx)
Figure 6—source data 3. Differential poly(A) tail length estimate results for vir-1 vs VIRc – Figure 6D.
Figure 6—source data 4. Poly(A) tail length estimates for CAB1 for reads from Col-0, vir-1 and VIRc – Figure 6D.

Figure 6.

Figure 6—figure supplement 1. Changes in the gene expression of circadian clock components in the vir-1 mutant.

Figure 6—figure supplement 1.

(A) Histogram showing log2 fold changes in gene expression based on Illumina RNAseq data for the vir-1 mutant and VIRc. Blue, genes with differential error rate sites (n = 5,095 genes); orange, genes without differential error rate sites (n = 14,686 genes). (B) Expression of core circadian clock components is perturbed in the vir-1 mutant. Boxplots showing normalized gene expression measured using Illumina RNAseq in log2counts per million (CPM): green, the VIR-complemented line (VIRc); orange, the vir-1 mutant. Asterisks, significant expression changes (using an FDR threshold of 0.05); orange labelled genes, genes with 3′ UTR m6A detectable by nanopore DRS and miCLIP. Each scatter point represents a single biological replicate. UBIQUITIN LIGASE 21 (UBC21; AT5G25760) was used as a control. (C) Expression of CCA1, encoding a regulator of the circadian rhythm in Arabidopsis, is increased in the vir-1 mutant. Boxplot showing the gene expression change from Col-0 (measured by RT-qPCR) for VIRc (green) and vir-1 (orange). Three technical replicates of three biological replicates were conducted. Each scatter point represents the comparison of a technical replicate of treatment (VIRc or vir-1) against control (Col-0). The expression change in vir-1 is significant (p = 0.02; marked by asterisk). UBIQUITIN LIGASE 21 (UBC21; AT5G25760) was used as a control. (D) Expression of the Flowering Locus C (FLC) gene is decreased in the vir-1 mutant. Boxplot showing gene expression change from Col-0 (measured by RT-qPCR) for VIRc (green) and vir-1 (orange). Three technical replicates of three biological replicates were conducted. Each scatter point represents the comparison of a technical replicate of treatment (VIRc or vir-1) against control (Col-0). Expression change in vir-1 is significant (p = 2.4 × 10−14; marked by asterisk). The following source data are available for Figure 6—figure supplement 1.
Figure 6—figure supplement 1—source data 1. Differential gene expression results for vir-1 vs VIRc, using counts derived from Illumina RNAseq – Figure 6—figure supplement 1A.
Figure 6—figure supplement 1—source data 2. qPCR expression of CCA1 and FLC in Col-0, vir-1 and VIRc – Figure 6—figure supplement 1C.