The Plant FYVE Domain-containing Protein FREE1 Associates with Microprocessor Components to repress miRNA Biogenesis (2022)
Figure 2

Figure 2. free1-ctmut causes a global increase in miRNA accumulation

A. sRNA sequencing analysis of miRNA expression in Col-0 and free1-ctmut mutants. X- and Y-axes indicate the logarithm base ten for miRNA expression in Col-0 and free1-ctmut. Compared to Col-0, miRNAs with at least 1.5-fold higher (free1-ctmut /Col-0 ≥ 1.5) or lower (Col-0/ free1-ctmut ≥ 1.5) expression in free1-ctmut are indicated by red and blue dots, respectively. The grey dots indicate differences in expression level < 1.5-fold (ratio < 1.5). The pie in the top left of the chart indicates the numbers of different categories of miRNAs. Data are derived from three biologically independent replicates.

B. Small RNA gel blots for 7-day-old seedlings Col-0 and free1-ctmut plants. U6 was used as a loading control. The numbers indicate the relative abundance of miR168, miR166, miR162, siRNA1003, tasiRNA255, IR71, miR156 and miR172 in the plants (the intensity in WT was arbitrarily annotated as 1.00).

C. Detection of the expression levels of miR156/160/165/166/396a target genes in WT and free1-ctmut plants. 7-d-old seedlings were used for RNA extraction followed by qRT-PCR analysis. UBQ10 was used as a reference. Data are presented as means ± s.d. (3 biological replicates). The asterisk indicates significant difference between the samples (*P < 0.05 and **P <0.01 by Student's t-test, compared with WT).

D. qRT-PCR analysis of pri-miR156/160/165/166/396a/396b in WT and free1-ctmut plants. 7-d-old seedlings were used for RNA extraction followed by qRT-PCR analysis. Data are presented as means ± s.d. (3 biological replicates). The asterisk indicates significant difference between the samples (*P < 0.05 and **P < 0.01 by Student's t-test, compared with WT).

E. Normalized counts per million mapped reads(CPM) of small RNAs mapped to miRNAs and miRNA*. Means ± s.d. for 3 biological replicates of wild type and free1-ctmut were indicated in the bars. The asterisk indicates significant difference between the samples (*P < 0.05 and **P < 0.01 by Student's t-test, compared with WT).

F. miRNA processing accuracy was calculated using all reads perfectly matched to the mature miRNA sequences versus all reads mapped to windows extending 20 nt on both sides of the mature miRNA sequence for the average of 3 biological replicates of wild type and free1-ctmut. The ratio was calculated only for those miRNAs that have at least 10 reads in each replicate. The values of minimum, first quartile, median, third quartile, and maximum for the boxplot were indicated. The data was statistically analyzed by Wilconxon test. No significant difference (P >0.05, n.s.) was indicated.

G. Ratio of miRNA/miRNA* between Col-0 and free1-ctmut mutants. The ratio was calculated using CPM values of the unique miRNAs and the sum of all the corresponding miRNA*s. The values of minimum, first quartile, median, third quartile, and maximum for the boxplot were indicated. The ratio was calculated only for those miRNAs that have reads for both miRNAs and their corresponding miRNA*s in all samples.