New class of microRNA targets containing simultaneous 5'-UTR and 3'-UTR interaction sites

Abstract

MicroRNAs (miRNAs) are known to post-transcriptionally regulate target mRNAs through the 3'-UTR, which interacts mainly with the 5'-end of miRNA in animals. Here we identify many endogenous motifs within human 5'-UTRs specific to the 3'-ends of miRNAs. The 3'-end of conserved miRNAs in particular has significant interaction sites in the human-enriched, less conserved 5'-UTR miRNA motifs, while human-specific miRNAs have significant interaction sites only in the conserved 5'-UTR motifs, implying both miRNA and 5'-UTR are actively evolving in response to each other. Additionally, many miRNAs with their 3'-end interaction sites in the 5'-UTRs turn out to simultaneously contain 5'-end interaction sites in the 3'-UTRs. Based on these findings we demonstrate combinatory interactions between a single miRNA and both end regions of an mRNA using model systems. We further show that genes exhibiting large-scale protein changes due to miRNA overexpression or deletion contain both UTR interaction sites predicted. We provide the predicted targets of this new miRNA target class, miBridge, as an efficient way to screen potential targets, especially for nonconserved miRNAs, since the target search space is reduced by an order of magnitude compared with the 3'-UTR alone. Efficacy is confirmed by showing SEC24D regulation with hsa-miR-605, a miRNA identified only in primate, opening the door to the study of nonconserved miRNAs. Finally, miRNAs (and associated proteins) involved in this new targeting class may prevent 40S ribosome scanning through the 5'-UTR and keep it from reaching the start-codon, preventing 60S association.

Figure 1.

Analysis of predicted interactions between 8-mers from different conservation classes and miRNAs. Closed bars indicate number of predicted interactions between 5′-UTR or 3′-UTR 8-mer sequences (indicated by 5U or 3U, respectively) and 5′- or 3′-ends (indicated by 5P or 3P, respectively) of a full set of mature miRNAs (A), of conserved miRNAs (B), and of nonconserved miRNAs (C). Open bars correspond to mean number of interactions after 1000 shuffling iterations, and error bars indicate standard deviations. (**) P < 5 × 10⁻⁵; (*) P < 5 × 10⁻³; (&) P < 0.05.

Figure 2.

Human miRNA hsa-miR-34a and target AXIN2. (A) Predicted interactions between hsa-miR-34a and AXIN2 UTR sequences. Extended seed match between the 5′-end of miR-34a and one of the 3′-UTR binding sites is shown in bold red. All predicted 3′-UTR sites are marked in the Supplemental material. Overlapping interactions between the 3′-end of miR-34a and the 5′-UTR inserted sequences are shown in bold blue. Energy was calculated using RNAhybrid. (B) Schematic showing vector constructs containing firefly luciferase reporter gene used in transfection experiments. The 5′-UTR and 3′-UTR inserts are indicated as 5U and 3U, respectively. (C) Luciferase expression fold change with miR-34a (red bars) normalized with negative control RNA oligo (blue bars). Firefly luciferase protein expression was normalized with Renilla luciferase protein. (D) Reporter constructs were co-transfected with anti-miR-34a oligo (red bars, Ambion, product ID, AM11030) and normalized with negative control RNA oligo (blue bars). (E) Effect of mutations in the 5′-UTR site—luciferase protein levels when reporter constructs were co-transfected with miR-34a (red bars) or negative control (blue bars). Error bars in panels C–E represent standard deviation from triplicate experiments.

Figure 3.

Human miRNA hsa-miR-34a and target WNT1. (A) Predicted interactions between hsa-miR-34a and WNT1 UTR sequences. Extended seed match between the 5′-end of miR-34a and the 3′-UTR binding site is shown in bold red. Interactions between the 3′-end of miR-34a and the 5′-UTR inserted sequences are shown in bold blue. (B) Schematic of vector constructs containing firefly luciferase reporter gene used in transfection experiments. 5′-UTR and 3′-UTR inserts are indicated as 5U and 3U, respectively. (C) Effect of 5′-UTR site in wild-type and mutant form on luciferase expression when treated with miR-34a. Renilla-normalized luciferase expression was normalized with negative control RNA oligo. Error bars represent standard deviation from triplicate experiments.

Figure 4.

Effect of 5′-UTR interaction site with lin-4-like on reporter expression levels. (A) Predicted interactions between lin-4-like and lin-28-like UTR sequences. The functional strand of the lin-4-like contains an intact cel-lin-4 seed region (red) while the 3′-end is modified (green). There is an extended seed match between the 5′-end of lin-4-like and the wild-type lin-28 3′-UTR binding site (bold red). The 3′-end of lin-4-like is complementary to the artificial lin-28-like 5′-UTR binding site created by introducing a few GC base pairs (bold italics) to form a perfect match. The wild-type lin-28 5′-UTR presents an imperfect match (bold blue). Structure and energy calculations were carried out using RNAhybrid. (B) Schematic showing vector constructs containing firefly luciferase reporter gene used in transfection experiments. lin-28-like 5′-UTR segments containing 8-mer perfectly matched and mutated sites are indicated as 5ULuc3U and 5UmutLuc3U, respectively. (C) Fold changes of Renilla-normalized firefly luciferase expression levels upon co-transfection with lin-4-like (red bars) with respect to nonspecific hsa-miR-16 (blue bars). Error bars represent standard deviation recorded from eight pooled replicates.

Figure 5.

Human miRNA hsa-miR-605 and SEC24D. (A) Predicted sites on the 5′-UTR and 3′-UTR targeted by the 3′-end and 5′-end, respectively, of hsa-miR-605. (B) Western blot analysis of SEC24D. Protein extract (40 μg) from three days post-transfected HeLa cells was separated by SDS-PAGE and probed with anti-SEC24D monoclonal antibody, with actin (ACTB) as control. (C) Densitometric analysis of the Western blots. X-ray films were scanned with HP Scanjet 3570c (Hewlett-Packard) and quantified using NIH image software. (D) SEC24D mRNA expression fold change in HeLa cells two days post-transfection with Negative Control-1, Pre-mir-605, or anti-miR-605. Error bars represent standard deviation from a triplicate experiment.