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Abstract
MicroRNAs (miRNAs) are endogenous small non-coding RNAs of ~23 nucleotides in length that form up a novel class of regulatory determinants, with a large set of target mRNAs postulated for every single miRNA. Thousands of miRNAs have been discovered so far, with hundreds of them shown to govern biological processes with impact on disease. However, very little is known about how they specifically interfere with biological pathways and disease mechanisms. To investigate this interaction, the hunt for direct miRNA targets that mediate the miRNA effects—the “needle in the haystack”—is an essential step. In this review we provide a comprehensive workflow of successfully applied methods starting from the identification of putative miRNA-target pairs, followed by validation of direct miRNA–mRNA interactions, and finally presenting methods that dissect the impact of particular miRNA-target pairs on a biological process or disease. This guide allows the way to be paved for obtaining biologically meaningful miRNA targets.
Highlights
In 1993, Victor Ambros and co-workers for the first time described a gene coding for small RNAs that are not translated, yet regulate another, protein-coding gene via base-pairing to its mRNA [1]
As even the original publication on let-7 described its binding to several distinct mRNAs, the broad spectrum of direct miRNA targets could be anticipated for the first time
The web interface provides the ability to look for miRNA recognition element (MRE) within the 5' UTRs and coding sequences of mRNAs, using the algorithm “miRWalk”
Summary
In 1993, Victor Ambros and co-workers for the first time described a gene coding for small RNAs that are not translated, yet regulate another, protein-coding gene via base-pairing to its mRNA [1]. At the core of this complex, the mature miRNA is physically associated with members of the Argonaute protein family (Ago 1–4 in human) [11], resulting in decreased protein output due to reduced translation, or degradation of the targeted mRNA. Available bioinformatic tools and databases which collect potentially direct or already validated direct miRNA targets will be discussed It will be outlined how high-throughput and low-throughput experiments—in combination with aforementioned in silico analyses—can aid in obtaining “high priority” target mRNAs. Third, the methodological possibilities to validate direct miRNA–mRNA interactions will be presented.
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