Figure 1

Prediction and detection of the let-7 miRNA–mediated target cleavage sites in the 3′UTR of TUSC2 mRNA.

(a) Cleaved mRNA 5′-fragments were detected by SLA–RT-PCR assay. Total RNAs were prepared from H1299 human lung cancer cells 48 h after treatment with or without the miR-98-LNA inhibitor (+/−miR-98-LNAi). RT was performed using SLA-RT primers specific to the 3′-end sequences of 5′-fragments of all the possible mRNA cleavage intermediates across the predicted let-7 miRNA:TUSC2 mRNA binding site (including seed, central bulge and supplementary pairing regions) and 3′- and 5′-adjacent regions, as depicted. The SLA-RT products were converted into cDNA and detected by subsequent PCR amplification. The specific SLA–RT-PCR products with predicted sizes ranging from 232 bp to 200 bp in the direction from 3′ to 5′ on target TUSC2 mRNA sequences were separated on 2% agarose gel (M: 0.03 μg of 1 kb DNA ladder) or quantified by qRT-PCR as demonstrated by the relative fragment abundance (RFA) on qRT-PCR histograms. The sequence-verified SLA–RT-PCR fragments are indicated by red arrows. The SLA–RT-PCR amplicon intensity with the predicted size in each lane represents the abundance of the potentially cleaved TUSC2 mRNA 5′-fragments with and without 3′-uridylation and was detected by the designated SLA-RT and 2U-SLA-RT primers, respectively (primer details in Supplementary Table 1). (b) Relative normalised expression (RNE) level of let-7 miRNA family members in H1299 cells treated or not treated with miR-98-LNAi was detected by real-time SLA–qRT-PCR. miR-622 and miR-30a were used as nonspecific controls and their expression levels were normalised to those of RNU44. SLA-RT primers and PCR primers are listed in Supplementary Table 3. (c) let-7 miRNA:TUSC2 target mRNA sequence pairing and potential cleavage sites were detected by a minimal free energy (MFE)–based miRmate algorithm¹⁷. The nt at the predicted and detected possible cleavage sites with the lowest MFE, cleavage blockage sites and mix of both are marked as red, blue and purple, respectively.