Abstract
Intracellular target identification of microRNA (miRNA), which is essential for understanding miRNA-involved cellular processes, is currently the most challenging task in miRNA-related studies. Although bioinformatic methods have been developed as the most efficient strategy for miRNA target identification, high-throughput experimental strategies are still highly demanded. In this review paper, we summarize and compare current experimental strategies for miRNA target identification, including gene expression profiling, immunoprecipitation and pull-down methods. Gene expression profiling methods mainly rely on the measurement of target gene expression through overexpression or inhibition of specific miRNA, which are indirect strategies to unveil miRNA targets. Immunoprecipitation methods use specific antibody to isolate RISC and bound mRNAs, followed by analysis with high-throughput techniques and bioinformatics to reveal miRNA-mRNA interactions. Pull-down methods use tagged miRNA mimics as probes to isolate associated target genes through affinity purification, which directly indicate miRNA-mRNA interactions after analysis of isolated target genes. Each method has its own advantages and limitations, which will be summarized and discussed in details. Overall, this review paper aims to provide a brief outline of recent achievements at experimental strategies for miRNA target identification. With the further development or improvement, we envision these experimental strategies will ultimately contribute a lot to the research on miRNA and miRNA-targeted biomedicine.
Highlights
MicroRNAs are endogenous small-noncoding RNAs with the length of ~ 22 nucleotides, which can regulate gene expression at the post-transcriptional level [1]
We introduce the general principles and applications of current experimental strategies for miRNA target identification
Due to the indirect isolation and identification, additional bioinformatic analysis are still needed to reveal miRNA-mRNA interactions from the Crosslinking and immunoprecipitation (CLIP) data [38, 39]. To address this issue, crosslinking, immunoprecipitation and sequencing of hybrids (CLASH) method, which is similar to CLIP but ligates miRNA and target mRNA in RNA-induced silencing complexes (RISC) together for further sequencing analysis, was developed (Fig. 3)
Summary
MicroRNAs (miRNAs) are endogenous small-noncoding RNAs with the length of ~ 22 nucleotides, which can regulate gene expression at the post-transcriptional level [1]. Sites in the 3’-UTR of target mRNAs that are complementary to miRNAs are usually known as “canonical” binding sites According to these information, bioinformatic methods based on different algorithms have been developed to predict miRNA target genes [15,16,17], which is the most commonly used strategy for exploring miRNA targets now. Recent studies revealed that “non-canonical” binding sites exist for some miRNAs to regulate their target mRNAs [19,20,21] and miRNAs could interact with non-coding RNAs [22], which cannot be predicted by bioinformatic methods either. Isolation methods based on immunoprecipitation of RISC with specific antibody and pull-down of miRNAmRNA complexes with different chemical tags have been developed. Gene expression profiling miRNAs function by regulating target gene expression through mediating mRNA degradation or inhibition of mRNA translation [1], indicating miRNA targets could be indirectly found out by quantifying expression changes of target genes including mRNAs and proteins
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