Figure 4

Effects of Target mRNA Structural and Positional Elements on Activities of miRNA-mediated Cleavage and Uridylation.

(a) The plasmid construct of pLJ-T214 containing the EGFP reporter gene with two copies of predicted let-7a target sequences in its 3′UTR. (b) Cleaved mRNA 5′-fragments from let-7 targets T1 and T2 were detected by SLA–RT-PCR (upper gel panels) and SLA-qRT-PCR (lower panels). The predicted sizes of SLA–RT-PCR products derived from target sites T1 and T2 were 240-209 bp and 532-500 bp, respectively. Total RNAs were prepared from H1299 at 16, 40 and 72 h after pLJ-T214 transfection and RT reactions were performed using SLA-RT or 2U-SLA-RT primers. The corresponding target T2 cleavage and uridylation activities detected by SLA-qRT-PCR with T2 fragment specific PCR primers and TaqMan probe. (c) The plasmid construct of pLJ-T722. The 3′UTR of the EGFP reporter gene containing two copies of identical predicted let-7:target pairing sequences but with varied lengths and compositions of nt (underlined) at their 5′- and 3′-adjacent regions (ST1 and ST2). (d) The effect of structural composition of target mRNA on let-7 miRNA–mediated mRNA cleavage activity as detected by SLA–RT-PCR assay (upper gel panels) and SLA-qRT-PCR (lower panels). Total RNAs were prepared from H1299 cells transfected with pLJ-T722 at 24 h and SLA–RT-PCR was performed using SLA-RT primers. D50, a PCR primer specific to the pLJ-T722 transcript, was designed to bond at every 50 bases along the target sequences toward its 5′ end. The base numbers correspond to those shown in (b) and escaped bases are indicated by “•” between them. M: 0.03 μg of 1 kb DNA ladder. A Taqman probe-based qPCR assay was used to detect cleavage activity specific to target ST2 and a SYBR Green-based qPCR to detect both target ST1 and ST2 cleavage activities, respectively. All qPCR results were presented as relative fragment abundance (RFA). Each RFA value was represented as the mean of three independent experiments and error bars as standard errors to the mean. The target cleavage and uridylation activities in miRNA target regions were highlighted in red boxes.