miRNAs (microRNAs) are small, single-stranded RNA molecules (19-24 nucleotides in length) that are generated from endogenous hairpin-forming transcripts encoded in the genomes of humans, animals, plants and viruses. Thus, miRNAs belong to the broad and constantly growing class of small non-coding RNAs including siRNA, stRNA, snoRNA and snRNA.
Surprisingly, miRNAs are one of the largest gene families, consisting of hundreds or, in higher organisms, more than a thousand genes. So far, the findings from the Human Genome Project and subsequent research efforts indicate that the human genome contains at least ~450 miRNAs. Various studies indicate that the human genome might contain up to 1500 miRNA genes.
miRNA biogenesis
miRNA genes have quite complex structures. They are usually transcribed by RNA Polymerase II into capped, polyadenylated and spliced transcripts. miRNA-containing sequences form one or more hairpin loop structures that are called primary miRNA structure (pri-miRNA). The RNase III Drosha-DGCR8 nuclear Microprocessor complex cleaves the base of the hairpin to form a precursor molecule called pre-miRNA (1). The pre-miRNA molecule is then actively transported out of the nucleus into the cytoplasm by Exportin 5 (Exp5), a carrier protein.
The Dicer enzyme (a key enzyme of the siRNA pathway) then cuts 20-25 nucleotides from the base of the hairpin to release the mature miRNA (2). When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNA molecules are formed, but only one is integrated into the RISC (RNA-Induced Silencing Complex). This strand is known as the guide strand and is selected by the argonaute protein, the catalytically active RNase in the RISC complex, on the basis of the stability of the 5’ end (3). The remaining strand, known as the anti-guide or passenger strand, is degraded as a RISC complex substrate (4) After integration into the active RISC complex, miRNAs base pair with partially complementary mRNA molecules and induce post-transcriptional down-regulation of the corresponding target protein by an unknown mechanism. It is as yet unclear how the activated RISC complex locates the mRNA targets in the cell, though it has been shown that the process is not coupled to ongoing protein translation from the mRNA (5).
miRNA function
The key role of miRNAs appears to be in gene expression regulation. For that purpose, miRNAs recognize partially complementary target sites into the 3’ UTR region of one or more messenger RNAs (mRNAs). In some cases, the annealing of the miRNA to the mRNA triggers the degradation of the mRNA transcript through a process similar to RNA interference (RNAi), but in most cases it is believed that the miRNA complex blocks the protein translation machinery or otherwise prevents protein translation without causing the mRNA to be degraded. Recent reports indicate that miRNAs might also control gene expression through chromation remodeling.
This effect was first described for the worm C. elegans in 1993 by R. C. Lee of Harvard University. As of 2002, miRNAs have been confirmed in various plants and animals, including C. elegans, human and the plant A. thaliana. Genes have also been found in bacteria that are similar in the sense that they control mRNA abundance or translation by binding an mRNA by base pairing, however they are not generally considered to be miRNAs because the Dicer enzyme is not involved.
miRNA regulation has a major impact on the proper regulation of a cell, and thus of the organism. Studies in which parts of the miRNA processing machinery have been knocked out indicate that an organism cannot survive in its absence. Less well known is the impact of individual miRNAs on their target genes. This is because target prediction is complicated. However, it is likely that miRNAs function similar to transcription factors. Their impact on target regulation can vary from minor to significant depending on a variety of factors. A report from May 2006 examined the level of control exerted by a miRNA specific for hematopoietic cells (6). The work indicated that a single miRNA could delineate gene expression between cells of hematopoietic and non-hematopoietic lineages in mice. This work offers indirect, but important proof of the potential regulatory impact a miRNA can have on gene regulation.
miRNAs and diseases: the missing link
There is a lot of interest in miRNAs in the scientific community. Since the early stages in the field, there has been speculation that miRNAs may be causative for human disease, including cancer.
Recent papers provide solid evidence that indeed, some miRNAs are oncogenic (7,8). By measuring the expression of 217 human miRNAs in cancer samples, Lu et al. found that the pattern of miRNA expression varies dramatically across tumour types. Interestingly, the expression pattern of this small set of miRNAs defines the cancer type better than expression data from 16000 mRNAs. As might be expected from the role of some miRNAs in development, the miRNA profiles of tumours are in accord with the tumours’ developmental history; tumours derived from tissues with a common embryonic precursor share similar miRNA expression patterns. Small RNAs can easily be measured from the formalin-fixed tissue specimens used routinely in hospital pathology laboratories; so potential miRNA-based diagnostics could fit simply into the standard hospital workflow.
This information provides an important missing link in our understanding of the molecular causes of many diseases including cancer. In the coming years, miRNA profiling could dramatically improve the diagnosis of poorly defined cancers with unknown origins, allowing better-informed choices for treatment. Finally, miRNA inhibitors could likely become a powerful class of anti-cancer therapeutic agents.
References
(1) DENLI AM, TOPS BB, PLASTERK BB, KETTING RF, HANNON GJ. (2004). Nature 432(7014):231-5.
(2) BERNSTEIN E, CAUDY AA, HAMMOND SM, HANNON GJ. (2001). Nature 409(6818):363-6.
(3) KURIHARA Y, WATANABE Y. (2004). Proc Natl Acad Sci USA 101(34):12753-8.
(4) PREALL JB, HE Z, GORRA JM, SONTHEIMER EJ. (2006). Curr Biol 16(5):530-5.
(5) GREGORY RI, CHENDRIMADA TP, COOCH N, SHIEKHATTAR R. (2005). Cell 123(4):631-40.
(6) SEN GL, WEHRMAN TS, BLAU HM. (2005). Differentiation 73(6):287-93.
(7) BROWN BD, VENNERI MA, ZINGALE A, SERGI LS, NALDINI L (2006). Nature Medicine 12 (5): 585-591. PMID 16633348. (8) O’DONNELL KA, WENTZEL EA, ZELLER KI, DANG CV, MENDELL JT (2005). Nature 435 (7043): 839-843. PMID 15944709.
(9) LU J, GETZ G, MISKA EA, ALVAREZ-SAAVEDRA E, LAMB J, PECK D, SWEET-CORDERO A, EBERT BL, MAK RH, FERRANDO AA, DOWNING JR, JACKS T, HORVITZ HR, GOLUB TR (2005) Nature 435 (7043): 834-838. PMID 15944708.
