MicroRNAs (miRNAs), which are approximately 21- nucleotide-long RNA regulators of gene expression1–3, have become a major focus of research in molecular biology. Although for a long time they were considered to be exclusive to multicellular organisms and possibly essential for the transition to a more complex organism design, the recent identification of miRNAs in the unicellular algae Chlamydomonas reinhardtii4,5 indicates that miRNAs are probably evolutionarily older than originally thought.
One to two hundred miRNAs are expressed in lower metazoans and plants, but at least a thousand are predicted to operate in humans. Functional studies indicate that miRNAs participate in the regulation of almost every cellular process investigated and that changes in their expression are observed in — and might underlie — human pathologies, including cancer1,2,6–8. These findings are perhaps not so surprising as bioinformatic predictions indicate that mammalian miRNAs can regulate ~30% of all protein-coding genes.
With just one possible exception noted so far9 , miRNAs control gene expression post-transcriptionally by regulating mRNA translation or stability in the cytoplasm10–14. However, further functions of miRNAs seem likely.
For example, by virtue of base pairing to RNA, miRNAs could regulate pre-mRNA processing in the nucleus or act as chaperones that modify mRNA structure or modulate mRNA–protein interactions. Indications that mammalian miRNAs can be imported into the nucleus15 or even secreted from the cell16 will motivate searches for currently unidentified functions for this class of molecule. What is already certain is that the discovery of miRNAs has revealed an important new dimension in the complexity of post-transcriptional regulation of eukaryotic gene expression.
We are beginning to understand why the 3′ UTRs of mRNA, with which the miRNAs and other factors interact, are often so long and so important for gene function. The mechanistic details of the function of miRNAs in repressing protein synthesis are still poorly understood. miRNAs can affect both the translation and stability of mRNAs, but the results from studies conducted in different systems and different laboratories have often been contradictory: a comprehensive and lucid picture of the mechanism of miRNA-mediated repression is difficult to elaborate.
In this Review, after briefly introducing miRNAs and their biogenesis, we summarize what is currently known about the mechanistic aspects of their function in controlling mRNA stability and translation, focusing primarily on animal cells. We also discuss the cellular localization and reversibility of miRNA-mediated repression. For further recent Reviews covering these topics, see Refs 11–14,17,18, and more general information about the biogenesis, diversity and function of miRNAs can be found in Refs 1–3,19–23.
miRNA and micro-ribonucleoprotein biogenesis miRNA precursors fold into imperfect dsRNA-like hairpins, from which miRNAs are excised in two steps, both of which are catalyzed by Drosha (also known as RN3) and the endoribonuclease Dicer — enzymes of the RNase III family (BOX 1).
The final processing of the ~70-nucleotide pre-miRNA hairpin by Dicer yields ~21-bp miRNA duplexes with protruding 2-nucleotide 3′ ends, similar to small interfering RNAs (siRNAs) operating in RNA interference (RNAi). Generally, the strand with the 5′ terminus located at the thermodynamically less-stable end of the duplex is selected to function as a mature miRNA, and the other strand is degraded3,19,21–23. miRNAs function as components of ribonucleoprotein (RNP) complexes or RNA-induced silencing complexes (RISCs), referred to as either micro-ribonucleoproteins (miRNPs) or miRNA-induced silencing complexes (miRISCs) (BOX 1). The most important and bestcharacterized components of miRNPs are proteins of the Argonaute family24,25. Mammals contain four Argonaute (AGO) proteins, AGO1 to AGO4. Their function in miRNA repression is demonstrated by their association with similar sets of miRNAs and their ability to repress protein synthesis when artificially tethered to the mRNA 3′ UTR26–28 (IG. 1). AGO2 is the only AGO that functions in RNAi because its RNaseH-like P-element induced wimpy testis (PIWI) domain, but not those of the other AGOs, can cleave mRNA at the centre of the siRNA–mRNA duplex (BOX 1). In Drosophila melanogaster, Argonaute1 is dedicated to the miRNA pathway, and Argonaute2 mainly functions in RNAi24,25. Apart from the AGO proteins, miRNPs often include other proteins, which probably function as miRNP assembly or regulatory factors, or as effectors mediating the repressive miRNP functions24.