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. 2021 Jan 13;185(4):1722–1744. doi: 10.1093/plphys/kiaa121

Figure 3.

Figure 3

A single-base substitution in the conserved complementary motifs unique to 3′-UTR of this family enhanced the URL1 mRNA stability. A, Position of the two conserved motifs CNS1 (19 nt) and CNS2 (21 nt) in the 3′-UTR of URL1. URL1-I, URL1-II, and URL1-III indicate the locations of primer sets used in (D). P1, P2, and P3 indicate the locations of primer sets used in (E). B, 5′-RLM-RACE showed that two putative cleavage products of about 300 and 100 bp (red triangle and pentagram) were present in both WT and Url1. Amplification of miRNA156 cleavage product of OsSPL14 was used as a positive control. C, The cleavage sites detected within 3′-UTR of URL1. Sixteen and 18 independent clones were sequenced for the large and small cleavage product, respectively. Arrows indicate cleavage sites relative to the CNS1 or CNS2 motif. D, RT-qPCR analysis of the URL1 mRNA transcript level in the Url1 mutant and WT using primer sets URL1-I, URL1-II, and URL1-III located in different regions of URL1, respectively. The rice ubiquitin gene was used as an internal control and the URL1 expression level in the Url1 mutant was normalized to the WT. Data are presented as mean ± se (n = 4). Significance of data is tested by Student’s t test (**P < 0.01). E, Quantification of nascent URL1 transcrips by biotin-16-UTP immunocapture nuclear run-on RT-qPCR in WT and Url1. RT-qPCR analysis were done with three primer sets P1, P2, and P3 which span the first, fourth, and fifth intron–exon bondary, respectively. F, The URL1 mRNA decay assay in the Url1 mutant and WT. URL1 mRNA level was determined by RT-qPCR analysis after treatment with cordycepin for 0, 1, 2, 3, 4, and 5 h. URL1 expression levels before cordycepin treatment (0 h) in WT and the Url1 mutant are set as 1.0, respectively. Data are presented as means ± se (n = 5). G, Schematic representation of 3′-UTR constructs used in the luciferase assay. H and I, Relative luciferase activities (H) and mRNA level (I) were measured when 3′-UTR, m3′-UTR, Δ3′-UTR of URL1 were inserted into the P35S::LUC reporter vector. Data are presented as mean ± se (n = 4), and the same letters above each bar indicate that means did not differ significantly at the 0.05 level in Tukey’s multiple comparison test.