microRNAs: A brief history of small molecules
Despite the central function that microRNAs appear to play in biological and disease processes, the first microRNA, lin-4, was only discovered in 1993. This was believed to be an anomaly until 2000 when a second microRNA, let-7, was described. Both microRNAs, identified from C. elegans, were highly unusual as their active transcripts were extremely small (~22nt) and were derived from hairpin structured RNA precursors. Unlike lin-4 however, the sequence of let-7 was found to be highly conserved in a wide range of organisms. It was soon realised that similar sequences, first termed microRNAs by Ambros in 2001, were widespread in the genomes of eukaryotes. Since this time, thousands of microRNAs have been cloned and characterised from a diverse range of organisms including arthropods, nematodes, platyhelminthes, vertebrates, plants and viruses. There are currently over 500 human microRNAs listed in the miRBase database (http://microrna.sanger.ac.uk/sequences/), accounting for about 1% of the human transcriptome, although it is predicted that the true figure is likely to be closer to one thousand.
Biosyntheisis
MicroRNAs are transcribed as 5’-capped large polyadenylated transcripts (pri-microRNA), primarily in a Pol II-dependent manner. Approximately 40% of human microRNAs are co-transcribed as clusters encoding up to eight distinct microRNA sequences in a single pri-microRNA transcript. Pri-microRNAs are cleaved within the nucleus by the microprocessor complex consisting of Drosha, an RNaseIII-type nuclease and a protein co-factor, DGCR8 (DiGeorge syndrome critical region 8 gene) in humans, Pasha in Drosophila. The resulting 60-70 nucleotide hairpin structure (pre-microRNA) encodes for a single microRNA sequence that is exported from the nucleus to the cytoplasm by Exportin5 in a Ran-GTP dependent manner. Cytoplasmic pre-microRNAs are further cleaved, by another RNaseIII-nuclease, Dicer in concert with cofactors (TRBP and PACT in humans), to remove the loop sequence forming a short-lived asymmetric duplex intermediate (microRNA: microRNA *). The microRNA: microRNA * duplex is in turn loaded into the miRISC complex in which Argonaut (Ago) proteins appear to be the key effector molecules. The strand that becomes the active mature microRNA appears to be dependent upon which has the lowest free energy 5’ end and the other strand is degraded by an unknown nuclease .
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Function
The consequence of miRISC-loaded microRNAs is largely dependent upon the degree of complimentarity between the microRNA and its target gene. Most plant microRNAs have perfect homology to their targets and their primary action appears to be the degradation of mRNA in a process similar if not identical to that found in the siRNA pathway. Although some animal microRNAs are believed to act in a similar fashion, nearly all animal microRNAs contain only limited sequence homology to their targets, usually limited to a 5’ 6-8 nt ‘seed region’, and act in a more subtle way to repress translation without mRNA degradation. How this occurs remains unclear. Early experiments with C.elegans suggested that repression occurs by prevention of elongation or degradation of nascent translation products. It has since been suggested that mRNA bound to the microRNA-miRISC complex may be sequestered away from the translational machinery in P-bodies that additionally act in concert with enzymes to remove the 5’-cap from mRNA hence preventing translation. Alternatively it has been suggested that microRNAs may prevent recognition of the 5’cap by translation factors.
Abnormal expression of microRNAs is commonly found in cancer
The potential importance of microRNAs in cancer is implied by the finding that the majority of human microRNAs are located at cancer-associated genomic regions, and in mice are frequently found near cancer susceptibility loci. The first paper to describe a link between aberrant microRNA expression and cancer was by Calin et al. in 2002 who found that two miRNAs, miR-15a and miR-16-1 encoded at the 13q14 locus, a region deleted in the majority of chronic lymphocytic leukemia (CLL) patients, were down-regulated in 68% of cases that harboured this deletion. Since this initial finding a plethora of research has arisen describing aberrant expression of microRNAs in a wide range of cancer types including most of the common solid and haematological malignancies. Moreover it has been suggested that microRNA expression profiling can distinguish cancers according to diagnosis and developmental stage of the tumour to a greater degree of accuracy than traditional gene expression analysis. MicroRNAs are proposed to play a direct role in oncogenesis as they can function both as oncogenes (e.g. miR-155 and members of miR-17-92 cluster) and tumour suppressor molecules (e.g. miR-15a and miR-16-1).