MicroRNAs: An emerging portrait
Fifteen years ago, no one had even heard of microRNAs. Not anymore: These small but abundant regulatory, non-coding RNAs - initially thought to be an oddity of nematode biology - appear to control gene expression in all animals, as well as in plants, some viruses, and at least one unicellular alga. So far, scientists have tracked their activity in all major organ systems, illustrated on the following pages.
Already, we see some general principles: Some animal micro-RNAs (miRNAs) are specific to a single tissue or cell type, others are expressed across multiple organs, and each organ seems to have a unique miRNA "profile" of characteristic expression levels among a set of miRNAs. These profiles change throughout development and during diseases such as cancer, suggesting that they may be useful diagnostic tools.
There's still a lot to learn. MiRNAs inhibit messenger RNA (mRNA) translation, but most miRNAs have not been confidently matched with mRNA targets. Their significance, however, is unquestionable. "Almost every important gene and pathway will be regulated at multiple levels by a variety of microRNAs," predicts Deepak Srivastava of the University of California, San Francisco. "It's really an entirely new layer of biology."
In the mouse inner ear, miRNAs exhibit a characteristic profile that appears during embryogenesis and remains into adulthood, suggesting that these miRNAs play a role in development and adult functioning.
Three of the miRNAs of the inner ear - miR-182, miR-183, and miR-96 (A) - are also present in sensory hair cells of zebrafish, indicating they have likely been evolutionarily conserved.
miRNA synthesis appears to be important for eye development in Drosophila melanogaster, as flies missing an miRNA-processing enzyme, Dicer, have abnormally small eyes whose components are either disorganized or missing entirely.
A cluster of three miRNAs is expressed in photoreceptors, bipolar cells, and amacrine cells in the retina (B), as well as in sensory hair cells in the inner ear. In vitro work shows that these miRNAs (miR-182, miR-183, and miR-96) target many transcription factors, including one important for retinal pigmented epithelium.
Fish, birds, and mammals have two miR-1 genes, miR-1-1 and miR-1-2, which encode identical miRNAs. According to the first report of an miRNA knockout mouse, published in Cell (129:303-17, 2007), deletion of miR-1-2 causes defects in many heart functions, including cardiac morphogenesis, electrical conduction, and cell-cycle control, even though miR-1-1 was still present. "That was very surprising to me," says Srivastava.
Four miRNAs appear to be present at lower levels in human breast cancer cells than in healthy breast tissue, and a combination of 15 miRNAs distinguishes cancerous from normal breast tissue with 100% accuracy (Cancer Res, 65:7065-70, 2005).
miRNAs appear to play a complex role in the development of both healthy and unhealthy lung cells. Genomic regions commonly deleted in lung cancer patients coincide with several genes encoding the miRNA let-7, and let-7 expression is decreased in lung cancer. Lung cancer patients with lower let-7 expression died sooner than those with higher let-7 expression. In mice, airways stop branching (C) in the developing lung if Dicer is inactivated, and this effect seems to be separate from Dicer's more general role in cell death.
Animals missing a single miRNA have widespread immunodeficiencies, according to two studies of miR-155 knockout mice published in April 2007 (Science, 316:604-11, 2007). miR-155 is required for normal function of B and T lymphocytes and dendritic cells. "There's no question that [miRNAs] are going to be important players in the control of the lymphocyte system," says Klaus Rajewsky of Harvard Medical School.
miRNAs also mediate interactions between viruses and host cells. Evidence suggests that viral RNAs are processed by mammalian miRNA machinery, creating viral miRNAs that then regulate host genes (D). For example, herpes simplex virus-1 inhibits apoptosis of infected neurons, which keeps the host cell alive.
miRNAs appear to be important for blood vessel formation, as genetic silencing of Dicer reduces human capillary sprouting in vitro and mouse angiogenesis in vivo. In human tissue, miRNAs likely promote blood vessel formation by suppressing genes that inhibit this process, such as thrombospondin.
miR-15 and miR-16 are found at a chromosomal region deleted in more than half of B cell chronic lymphocytic leukemias (B-CLL). miRNA is expressed differently in CLL samples versus normal B cells.
Some of the earliest evidence of a microRNA role in organogenesis came from studies showing that Caenorhabditis elegans deficient in the miRNA let-7 die by bursting at the vulva (E).
Expression of several miRNAs is altered in colorectal cancer, and one of them, miR-31, may correlate with the stage of cancer progression (E).
Disruptions in miRNA-processing enzymes Dicer or Drosha in C. elegans provided the first clue that microRNAs are important in the germline. Recent work (Nature, published online Aug. 29, 2007) found that two miRNAs are important for setting up the basic body plan after fertilization. In the mouse, oocytes without functional Dicer have problems with chromosomal movements and spindle arrangement, and these cells arrest during meiosis.
Most miRNA research in the liver revolves around miR-122, which appears to be both liver-specific and the most common miRNA in the adult mammalian liver. Research suggests miR-122 is involved in plasma cholesterol levels and fatty acid concentration. miR-122 also enhances replication of hepatitis C virus RNA in cultured human liver cells, probably by binding directly to viral RNA.
One miRNA, miR-375, appears important for insulin regulation in pancreatic ?-cells (F). In one paper, inhibited insulin secretion in mouse islet cells was reported (Nature, 432:226-30, 2004).
As in other cancers, pancreatic cancer tissue shows a distinct miRNA profile: Expression of 25 miRNAs can differentiate pancreatic cancer from normal pancreatic tissue in 90% of samples, while high expression of one miRNA consistently predicts poor survival (JAMA, 297:1901-8, 2007).
miR-140 is specifically expressed in the cartilage of mouse embryos during long bone and flat bone development, including in the digits, sternum, ribs, vertebral column, and the cartilage base of the skull.
Loss of Dicer in mice disrupts hair follicles and is associated with decreased proliferation of follicular cells and increased proliferation of epidermal cells. Emerging research (DNA Cell Biol, 26:227-37, 2007) suggests that miRNAs might be important for multiple aspects of wound healing, including regrowth of blood vessels. (G) miRNAs seem to modulate vascular endothelial growth factor (VEGF) signaling.
Removal of Dicer in mice results in both massive cell death in the limb and dysregulation of limb-specific genes.
Muscle-specific miR-1 is one of the most evolutionarily conserved miRNA families and is highly expressed in both progenitors and adult muscle. A paper in Nature Genetics (38:813-8, 2006) showed that a mutation that creates a target site for miR-1 produces abnormally muscular sheep. miR-181 is highly expressed during muscle differentiation but expressed at very low levels in adult muscle, showing that miRNAs can be important for tissue differentiation even if they're not active in the mature tissue.
miRNAs seem to be particularly important for nervous system development: Many are differentially expressed during mammalian brain development and are found in specific regions of the brain.
miRNAs appear to regulate major events during neurogenesis (H). For example, flies that lack miR-9a, a miRNA specific to the nervous system in several species, develop extra sense organs.
Recent evidence suggests that miRNAs may also be involved in adult brain function, specifically in the synaptic remodeling that underlies memory formation. In 2006, researchers reported (Nature, 439:283-9, 2006) that miR-134, a brain-specific miRNA that is highly conserved among species, inhibits synthesis of dendritic proteins in rat hippocampal neurons, probably by repressing a kinase that builds dendritic spines. They also found that stimulating neurons with brain-derived neurotrophic factor relieved this suppression, hinting that chemical changes induced by learning and memory may control synaptic growth through miR-134. An August 2007 study from Ken Kosik's lab at the University of California, Santa Barbara (see "Seafloor to benchtop,") also discovered (RNA, 13:1224-34, 2007) that many other miRNAs accumulate in rat dendrites.
Very recent work has provided evidence of miRNA involvement in circadian rhythms and sleep, as well as in neuropsychiatric diseases such as Fragile X syndrome, Tourette syndrome, and other forms of neurodegeneration.
miR-375, which regulates insulin secretion in the pancreas, is also highly expressed in the pituitary gland of zebrafish embryos, suggesting that it may regulate secretion of other hormones. miRNA expression profiles distinguish healthy tissue from noncancerous pituitary tumors and between samples taken from treated and untreated patients.