To address these problems, several labs have utilized high-throughput sequencing following ultraviolet (UV) crosslinking and immunoprecipitation (CLIP-seq or HITS-CLIP) and other similar methods to identify Argonaute/miRNA binding sites. Furthermore, there is little consensus among miRNA target prediction programs. However, these programs often miss non-canonical target sites and have high rates of false positives, as the presence of a binding site does not ensure regulation. In silico approaches are often based on common motifs, such as the “seed” sequence found in nucleotides 2–7 of the miRNA, and the propensity for miRNAs to bind to the 3'UTR of messenger RNAs. In these cases, as few as 7 nts can provide functional targeting information to the miRISC. Predicting targets is complicated by the limited sequence information provided by miRNAs due to their small size and their ability to function even when only a partial duplex is formed. Over the past decade, one of the greatest challenges in the field has been identifying miRNA target sites. More recently, it has been shown that miRNAs can target regions throughout mRNAs and non-coding RNAs. Classically, miRNAs are thought to help direct the miRISC to the 3'UTR of messenger RNAs (mRNAs), resulting in a decrease in gene expression through mRNA destabilization and/or translational repression. Mature miRNAs are loaded onto an Argonaute (Ago) protein as part of the miRNA Induced Silencing Complex (miRISC). Maturation of the miRNA occurs after subsequent Dicer processing of the pre-miRNA into its final ~21 nt form. This long, poly-adenylated transcript is capable of forming an imperfectly paired stem-loop structure and is processed by Drosha and DGCR8/Pasha into a ~65 nucleotide (nt) long hairpin precursor-miRNA (pre-miRNA). The majority of miRNA biogenesis begins with transcription of a primary-miRNA (pri-miRNA) transcript by RNA polymerase II. As a consequence of their broad regulatory potential, altered miRNA expression is implicated in many diseases.

This flexibility in targeting allows each miRNA to potentially regulate hundreds of different transcripts in a range of pathways. In many eukaryotic organisms, small RNA molecules, known as microRNAs (miRNAs), provide post-transcriptional control of gene expression through imperfect binding to target RNAs.

Iclip protocol