The seminal discoveries regarding microRNA and their importance in gene regulation began rather quietly. While working together in the 1980s as postdoctoral research fellows at the Massachusetts Institute of Technology (MIT), molecular biologists Victor Ambros and Gary Ruvkun were studying two gene mutations in the nematode Caenorhabditis elegans that had opposite effects on developing cells. One, in the gene lin-4, kept the worm’s larvae from developing into fully formed animals. The other, in the gene lin-14, caused the worms to skip early developmental stages and mature prematurely. It appeared that lin-4 repressed lin-14, but the molecular mechanism for that repression remained a mystery.
In the early 1990s, while in his own lab at Harvard University, Ambros and his colleagues successfully isolated and cloned lin-4. They had expected that the gene’s product would be a standard regulatory protein, but instead it turned out be a tiny non-protein-coding strand of RNA about 22 nucleotides long. This was the first identification of what would later be called a microRNA, although the significance of the finding would not become clear for several more years.
Around the same time, Ruvkun, who was at Harvard University, had cloned the lin-14 gene and identified the specific area on it—the 3’ untranslated region (3’ UTR)—that is highly conserved in the homologue of the lin-14 gene in other nematodes and targeted by lin-4. When Ambros and Ruvkun compared the lin-4 and lin-14 sequences, they discovered that the 22-nucleotide lin-4 RNA and the 3’ UTR were partially complementary, which suggested that lin-4 was regulating lin-14 by binding to these shared sequences. The complementary sequences were also on evolutionarily conserved areas of the genes, which meant it was unlikely that this finding was a statistical fluke.
In 1993, Ambros and Ruvkun published back-to-back papers about their findings in the journal Cell. Ambros described how the regulatory product of lin-4 was a 22-nucleotide-long RNA, and Ruvkun reported on how that tiny RNA was able to suppress the messenger RNA (mRNA) expressed by lin-14 by binding directly to the mRNA itself. Ruvkun showed that the mechanism by which the lin-4 microRNA represses the lin-14 mRNA is at the level of translation of this mRNA into protein. The molecular mechanism by which lin-4 repressed lin-14 and thereby advanced C. elegans larval development had now been explained.
At the time, however, it was suspected that the involvement of microRNAs in gene silencing might be restricted to C. elegans, as it was apparently the only organism with lin-4 in its genetic makeup. But then, in 1999, British plant biologist David Baulcombe identified, for the first time, small RNAs that mediate a similar silencing process in plants. A year later, Ambros’s lab discovered that lin-4 regulated another gene in C. elegans, lin-28—a finding that suggested that microRNA might be functioning throughout the C. elegans genome. Then, in 2000, Ruvkun announced the discovery of a second C. elegans microRNA, let-7. And in a dramatic advance in 2000, Ruvkun showed that let-7 was evolutionarily conserved across the animal kingdom, including in humans. The conservation of the let-7 RNA argued for the generality of microRNAs. It was now clear that microRNAs and their potential for repressing gene expression were not just a peculiarity of one species of worm.
Today, microRNAs are recognized as playing a major role in gene expression, and their discovery has launched an exciting new field of scientific research. Scientists have linked the gene-repressing abilities of microRNAs and small interfering RNAs to a diverse range of important developmental and physiological processes in both plants and animals. MicroRNAs in the human genome (which may number as many as 1,000) have been found to play key roles in both health and disease, including embryonic development, blood-cell specialization, muscle function, heart disease, cancer and viral infections.