TargetScan primarily predicts potential miRNA targets by looking for the presence of 8mer, 7mer, and 6mer sites that match the seed region of every miRNA. It is possible to choose to include only conserved sites. Other features may be evaluated, such as seed-pairing stability, 3' compensatory pairing, and target site abundance. The putative targets are then ranked based on the predicted efficacy of targeting, calculated using cumulative scores considering all the features of the sites. Moreover, TargetScan predicts the secondary structure to evaluate the free energy of predicted complexes.

TargetScan is a software that is very effective for predicting miRNA binding sites in mammals. Before predicting miRNA target genes, the 3' UTR region of the transcript needs to be determined. TargetScan database identifies the transcript's corresponding 3' UTR region through a sequencing technique called 3P-seq (miRNAs in mammals have a post-transcriptional regulatory role by binding the 3' UTR region of the transcript sequence). Since the 3' UTR annotations are already available in NCBIT, the TargetScan database provides a comprehensive 3' UTR region sequence prediction in combination with the analysis.

Fig.1 TargetScan software

MiRNAs are conserved across species. By predicting miRNA target sites in different species, the TargetScan team found that their binding sites are also conserved and can be divided into the following categories:

Fig.2 TargetScan team found that their binding sites can be divided into these categories

Furthermore, they are divided into different miRNA families according to the specific binding regions and the results of multiple sequence alignment of miRNAs. MiRNA family are categorized as “broadly”, “poorly”, or “conserved” based on the conservation of MiRNA sequences among different species.

Fig.3 TargetScan software in mammals

To search predicted microRNA targets in mammals, we used a library of sub links:

• https://www.targetscan.org/mmu_80/
• https://www.targetscan.org/vert_80/
• https://www.targetscan.org/worm_52/
• https://www.targetscan.org/fly_72/
• https://www.targetscan.org/fish_62/

The TargetScan Search Function

TargetScan uses either the name of the gene to be predicted or the ENST number to display all the miRNA loci predicted for that gene. Let's take miR-3074-5p as an example. Once you have entered it, click Submit.

Fig.4 Take miR-3074-5p as an example to search its target gene

Fig.5 Target gene display for miR-3074-5p

Here are the predicted results. Colored dots are marked with predicted targets. Targetscan 7 uses a 3'-UTR configuration file. The above 3'-UTR map is constructed using a 3P-seq tag (the number of 3' UTR tags marked on the y-axis), indicating the location and usage of mRNA cleavage and Polyadenylation sites. 3P-seq tags from multiple cell lines or tissues were normalized to each other (to account for variable sequencing depths) and then pooled into a set of consensus counts. We assigned the normalized 3P-seq tag to the representative 3' UTR of each stop codon and add them together (as shown on the left side of the configuration file) to quantify the usage of the stop codon. This also includes five pseudo-counts added remotely to the GENCODE comments. Comments for each representative 3' UTR start with the longest GENCODE 3' UTR and are sometimes extended with information from 3P-seq or other comment sources. The 3'-UTR profile (red line) drops with the 3P-seq tag for each cluster, indicating the proportion of transcripts containing the 3' UTR fragment. Each TargetScan 3'-UTR profile also displays the farthest location of the GENCODE annotation (blue vertical line with Ensembl record ID). Each prediction result can be clicked (each small color box). When clicked, the information in the area below will be updated.

The bottom shows the sequence around the selected miRNA target, and the left shows the corresponding site in other species. At the same time, we can see the position of the miRNA seed sequence in UTR, and the conserved seed sequence region will be highlighted in white. This is the detailed information on the locus, including the position of the miRNA seed region in this region, the sequence in the middle of the locus, the locus type, and the locus score. The lower the context + + score, the greater the probability that the locus is a target. In addition, percentile is the conversion of the score. The closer the score is to 100, the greater the probability that the locus is a real target.

Fig.6 The predicted binding sites for miR-3074-5p

PART2
Conserved.
This section shows the predicted consequential pairing of the target region (top) and MiRNA (bottom), the type of pairing, the context+ + score, and the percentage of the predicted consequential pairing of the target region (top) and MiRNA (bottom).

Fig.7 TargetScan predicts 8mer sites

7mer-m8: a perfect match to the 2-8(seed + 8) of the mature MiRNA
7mer-A1: a perfect match to the 2-7(seed) of the mature miRNA, followed by“A”
Clicking on Context + + score will result in 14 functional dimensions:

Fig.8 TargetScan: Context++ score contributions

Total context + + score represents the sum of all the scores of this MiRNA. The smaller the sum, the more likely the miRNA is to target the protein. Aggregate PCT represents the conservation of miRNA loci in the population.

In summary, miRNAs are post-transcriptional regulators of gene expression in both animals and plants. By pairing to microRNA responsive elements (mREs) on target mRNAs, miRNAs play gene-regulatory roles, producing remarkable changes in several physiological and pathological processes. Thus, the identification of miRNA-mRNA target interactions is fundamental for discovering the regulatory network governed by miRNAs. The best way to achieve this goal is usually by computational prediction followed by experimental validation of these miRNA-mRNA interactions, such as a dual luciferase reporter assay.

Fig.9 Schematic diagram of miRNA prediction after selection of the best model

Prove Interaction between miRNA and a Specific mRNA Target Site

When co-expression is verified, it is necessary to investigate the physical interaction between the miRNA under consideration and the candidate mRNA localized within the target mRNA. The reporter gene assay is the current gold standard procedure to prove a direct miRNA-mRNA interaction. In this procedure, the 3'-UTR regions of the gene of interest, containing the predicted mRNA sequence, are cloned immediately downstream of the luciferase gene or another reporter gene contained in a plasmid. Then, the plasmid must be subcloned under the control of a ubiquitous promoter. Subsequently, plasmid-containing cells can be co-transfected with miRNA mimics or miRNA inhibitors in order to perform gain-/loss-of-function experiments. The rationale for performing the luciferase assay is based on the evidence that though the mRNA is a real target of miRNA under examination, miRNA mimics, as well as miRNA inhibitors, are able to alter endogenous miRNA concentrations and can potentially lead to changes at protein levels.

Demonstrate miRNA-Mediated Effects on Target Protein Expression

After transfection of either miRNA mimics or inhibitors into the cells, protein expression needs to be analyzed. Protein changes can be detected by conventional procedures such as Western blotting, ELISA, and immunecytochemistry experiments.

If a given mRNA is a real miRNA target, many changes in levels of proteins should be detected as a consequence of an interaction between them. An increased miRNA activity, deriving from the transfection of miRNA mimic into cells expressing the target protein, should decrease target protein expression. On the other hand, a reduced miRNA activity due to the use of a miRNA inhibitor for cell transfection should result in increased target protein expression.

Various Designs Exist for miRNA Inhibitors

Locked-nucleic acid (LNA) oligonucleotides are synthetic, modified antisense RNAs. When introduced into cells, these single-stranded molecules perfectly bind to endogenous miRNAs, preventing hybridization with their cellular mRNA targets and thus decreasing miRNA activity. Similarly, constructs known as 'sponge' inhibitors produce RNA sequences containing several sites specific for miRNA in order to sequester endogenous miRNAs and subsequently inhibit their regulatory capacity.

In this method, appropriate controls need to be employed to determine transfection efficiency. For example, the transfection of scrambled miRNA sequences should prove the specificity of a miRNA-mRNA interaction, and the use of an empty vector should demonstrate the efficiency of the transfection.

Reference

1. Agarwal V, Bell GW, Nam J, Bartel DP. Predicting effective microRNA target sites in mammalian mRNAs. eLife, 2015; 4:e05005
2. Riolo G, Cantara S, Marzocchi C, Ricci C. miRNA Targets: From Prediction Tools to Experimental Validation. Methods Protoc. 2020; Dec 24;4(1):1.
3. Nicolas F.E. Experimental validation of microRNA targets using a luciferase reporter system. Methods Mol. Biol. 2011; 732:139-152.
4. Huang Y., Zou Q., Song H., Song F., Wang L., Zhang G., Shen X. A study of miRNAs targets prediction and experimental validation. Protein Cell. 2010; 1:979-986.