In the last few decades, we have been focused on genes that encoded proteins. Indeed, based on the genetic code, pieces of the genome that encode proteins are relatively easy to find. However the discovery of microRNAs as well as other RNAs that do not encode for proteins, indicates that there is much more to the genome than protein coding genes. Indeed, microRNAs represent ~4% of the genes in the human genome. This discovery suggests that the genome is far from being deciphered, and most importantly that miRNAs are likely to represent just the “tip of the iceberg” with many other small non-coding RNAs to be discovered. Proof of this is the recent identification of piwi-interacting RNAs (piRNAs), endogenous siRNAs, and intron derived miRNAs (miRtrons). In summary, the recent identification of novel regulatory RNAs has opened a new window to an important area of Biology that was unexplored until now, but has important implications in human development and disease.

What are microRNAs?

        MicroRNAs (miRNAs) are small non-protein coding genes present in virtually all animals and  plants. and tend to be transcribed from several different loci in the genome. These genes encode for long RNAs with a hairpin structure that when processed by a series of RNaseIII enzymes (Drosha and Dicer) form a miRNA duplex of ~22 nt long with 2nt overhangs on the 3’end. One strand of the duplex is incorporated in a protein complex that includes a member of the Argonaute family of proteins.

How to identify miRNA target mRNAs?

Three main experimental approaches have been used to identify miRNA targets. First, because miRNAs repress translation and accelerate target mRNA deadenylation, Microarrays have been used to identify mRNAs that are upregulated in the absence of miRNAs. Second, mass-spect approaches have been used to identify proteins upregulated in the absence of miRNAs. Finally, based on the idea that miRNAs must bind their targets, immuno precipitation of the miRISC complex and mRNA sequencing have been used to identify mRNAs that bound to miRISC. These approaches identify on the order of several hundred targets/miRNA. One of the biggest challenges at the moment is to identify which of these miRNA-Target interactions are indeed physiologically relevant. In the last few years we have developed target protectors. These target protectors that bind one of the targets specifically and allow us to interfere with the regulation of one target while all the other targets are still being regulated by the miRNA. These are modified oligonucleotides that bind the miRNA target site and compete with the miRNA for the binding to the target. Using these tools we have shown that miR-430 regulate the nodal pathway during gastrulation. We are currently using these approaches to dissect the functional relevance of individual miRNA target interactions during brain morphogenesis, angiogenesis and gastrulation.

What determines the recognition of a target mRNA by a microRNA?

        miRNAs only need to pair partially to the target mRNA in order to elicit translational repression. The partial complementarity between the target and the mRNA has made it difficult to identify the targets for every miRNA. Yet, through the work of several laboratories including Eric Lai, Victor Ambros, Gary Ruvkum, Frank Slack, Steve Cohen, Dave Bartel and Phil Sharp among others, have established a set of rules that highlight the seed of the miRNA as an important feature in the target recognition.

         The seed has been defined as the nucleotides 2-8 of the miRNA. Based on the examples studies up to date, a large fraction of the targets present in their 3’UTRs perfect Watson-Crick complementary sites to the seed of the miRNA. While this is not an universal rule, it is one of the best features that describes miRNA targets, yet there are many other features, such as low GC content around the seed, and the preferential positioning of the target site towards the edges of the 3’UTRs. Yet, not all the mRNAs with sites complementary to the seed are indeed miRNA targets, thus suggesting that there are additional factors that determine which genes are in fact under the regulation of miRNAs.

The miRNA serves as a guide for this complex (miRISC, miRNA Induced Silencing Complex) to the target mRNA where it induces translational repression and accelerates mRNA deadenylation. One major effector/interactor of the RISC complex is the protein GW182, which might serve as a docking protein to recruit additional effectors that trigger deadenylation (CCR4/NOT) and translational repression.