Micro RNA

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In genetics, micro RNAs (miRNA) are a form of single -stranded RNA that is typically 21-23 nucleotides long and whose main biological function is thought to be regulation the expression of other genes. miRNAs are specified by genes which are transcribed from DNA into an RNA called primary-micro RNA, (pri-miRNA), but are not translated into protein. Although the concept of miRNA is relatively recent, it is of major importance for understanding genetics, cell biology and evolution of genomes. In Drosophila flies there are estimated to be 8,000–12,000 miRNA target sites genome-wide, approaching 1 per gene [1][2]. In humans possibly more that 10 percent of genes contain a target site for a miRNA [3], indicating that a significant fraction of all animal genes are regulated by miRNA.


Formation of primary micro RNA (pri-miRNA) by transcription involves harpin loop formation

The DNA locus that specifies a miRNA is longer than the miRNA, and this DNA region includes both the miRNA sequence plus an approximate reverse complement sequence. (A reverse complement is a region with complementary bases in the reverse order, going from 5'-> 3' on the same DNA strand). When this DNA locus is transcribed into a single-stranded RNA (dsRNA) molecule, the miRNA sequence and its reverse-complement base pair to form a double stranded RNA hairpin loop; this forms a primary miRNA structure (pri-miRNA) which is the first stage in a series of events that occur in cells by which miRNAs affect cell functions. The specific details of these effects differ significantly in mechanistic detail between plants and animals.

Pre-miRNA formation from pri-miRNA in animals, Dicer in plants and animals

In animals, the nuclear enzyme Drosha cleaves the base of the hairpin in pri-miRNA to form pre-miRNA. The pre-miRNA molecule is then actively transported out of the nucleus into the cytoplasm by Exportin 5, a carrier protein. The Dicer enzyme then cuts 20-25 nucleotides from the base of the hairpin to release the mature miRNA. In plants, which lack Drosha homologues, pri- and pre-miRNA processing by Dicer probably takes place in the nucleus, and mature miRNA duplexes are exported to the cytosol by Exportin 5.

Major discoveries about miRNA

The function of miRNAs appears to be primarily in gene regulation, and for that purpose the complementarity of a miRNA to a part of one or more messenger RNAs (mRNAs) is important.

Animal miRNAs are usually complementary to a site in the 3' untranslated region (3' UTR) of a mRNA whereas plant miRNAs are usually complementary to coding regions of mRNAs. The annealing of the miRNA to the mRNA then inhibits protein translation, but sometimes facilitates cleavage of the mRNA. This is thought to be the primary mode of action of plant miRNAs. In such cases, the formation of the double-stranded RNA through the binding of the miRNA triggers the degradation of the mRNA transcript through a process similar to RNA interference (RNAi), though in other 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. miRNAs may also target methylation of genomic sites which correspond to targeted mRNAs. miRNAs function in association with a complement of proteins collectively termed the miRNP.

This effect was first described for the worm Caenorhabditis 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 Arabidopsis thaliana. Genes have 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. A key 1998 paper by Andrew Fire, Craig Mello and others (for which Fire and Mello were awarded a Nobel Prize in 2006) greatly stimulated research on miRNA.

They found potent and specific interference with gene expression when double stranded RNA (dsRNA) was injected into the worm. C. elegans. The expression of mRNA for a gene was silenced (with mRNA production of the target eliminated or greatly reduced) when dsRNA corresponding to the coding region of a gene was injected into the worm. Many different cells of the worm were affected, even progeny of the injected worm. The potency of the injected and ability of dsRNA to cross cell boundaries suggested to Fire and Mello that cells have specific uptake mechanisms for dsRNA and that entry of a few dsRNA molecules into a cell can silence genes possessing closely similar DNA sequences in their protein coding exons [4].

The term miRNA was first introduced 2001 in a set of three articles in Science[5].

In plants, similar RNA species termed short-interfering RNAs siRNAs are used to prevent the transcription of viral RNA. While this siRNA is double-stranded, the mechanism seems to be closely related to that of miRNA, especially taking the hairpin structures into account. siRNAs are also used to regulate cellular genes, as miRNAs do.

The activity of an miRNA can be experimentally blocked using a locked nucleic acid oligo, a Morpholino oligo[6] or a 2'-O-methyl RNA oligo. [7]Most efficient methods for miRNA detection are based on oligonucleotides modified with locked nucleic acids.[8]

microRNA regulation

microRNA regulation has a major impact on the proper regulation of a cell, and thus of the organism. Studies in which parts of the microRNA processing machinery have been knocked out indicate that an organism can not survive in its absence. Less well known is the impact of individual microRNAs on their target genes. This is because target prediction is complicated. However, it is likely that microRNAs 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 microRNA specific for hematopoietic cells [9]. The work indicated that a single microRNA 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 microRNA can have on gene regulation.

miRNA and cancer

miRNA has been found to have links with some types of cancer.

A study of mice altered to produce excess c-myc — a protein implicated in several cancers — shows that miRNA has an effect on the development of cancer. Mice that were engineered to produce a surplus of types of miRNA found in lymphoma cells developed the disease within 50 days and died two weeks later. In contrast, mice without the surplus miRNA lived over 100 days.[10]

Another study found that two types of miRNA inhibit the E2F1 protein, which regulates cell proliferation. miRNA appears to bind to messenger RNA before it can be translated to proteins that switch genes on and off.[11]

By measuring activity among 217 genes encoding miRNA, patterns of gene activity that can distinguish types of cancers can be discerned. miRNA signatures may enable classification of cancer. This will allow doctors to determine the original tissue type which spawned a cancer and to be able to target a treatment course based on the original tissue type. miRNA profiling has already been able to determine whether patients with chronic lymphocytic leukemia had slow growing or aggressive forms of the cancer.[12]


Cell-to-cell transfer of miRNAs

A recent study raises the possibility that one cell might regulate gene expression in another cell by transfer of miRNAs via vesicles, so-called exosomes, released into the extracellular fluid. [13]  Exosome-mediated transfer of miRNAs represents a novel method of cell-to-cell communication.

References

Citations

  1. http://biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pbio.0020396 (2004) MicroRNA Is a Major Regulator. PLoS Biol 2(11): e396 DOI: 10.1371/journal.pbio.0020396
  2. http://biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371%2Fjournal.pbio.0030085 Brennecke J, Stark A, Russell RB, Cohen SM (2005) Principles of MicroRNA–Target Recognition. PLoS Biol 3(3): e85 DOI: 10.1371/journal.pbio.0030085
  3. http://biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pbio.0020363 John B, Enright AJ, Aravin A, Tuschl T, Sander C, et al. (2004) Human MicroRNA Targets. PLoS Biol 2(11): e363 DOI: 10.1371/journal.pbio.0020363
  4. Fire, S. Xu, MK Montgomery, SA Kostas, SE Driver and Mello, CC (2004). "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans". Nature 391 (19 February 1998): 806-811. PMID 9486653.
  5. Ruvkun, G. (Oct 26 2001). "Molecular biology. Glimpses of a tiny RNA world.". Science 294 (5543): 797-9. PMID: 11679654.
  6. Kloosterman, WP; Wienholds E, Ketting RF, Plasterk RH (Dec 7 2004). "Substrate requirements for let-7 function in the developing zebrafish embryo". Nucleic Acids Res. 32 (21): 6284-91. PMID: 15585662.
  7. Meister, G; Landthaler M, Dorsett Y, Tuschl T (Mar 2004). "Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing". RNA 10 (3): 544-50. PMID: 14970398.
  8. Kloosterman, WP; Wienholds E, de Bruijn E, Kauppinen S, Plasterk RH (Jan 2006). "In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes". Nat Methods (1): 27-9. PMID: 16369549.
  9. Brown BD, Venneri MA, Zingale A, Sergi LS, Naldini L (2006). "Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer". Nature Medicine 12 (5): 585-591. PMID 16633348.
  10. He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Goodson S, Powers S, Cordon-Cardo C, Lowe SW, Hannon GJ, Hammond SM (2005). "A microRNA polycistron as a potential human oncogene". Nature 435 (7043): 828-833. PMID 15944707.
  11. O'Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT (2005). "c-Myc-regulated microRNAs modulate E2F1 expression". Nature 435 (7043): 839-843. PMID 15944709.
  12. 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). "MicroRNA expression profiles classify human cancers". Nature 435 (7043): 834-838. PMID 15944708.
  13. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol advanced online publication

Further reading

The first two papers describe the way in which microRNAs were plus discovered plus historically fascinating details of the failures of the investigators:
  • Lee R, Feinbaum R, Ambros V. (2004). "A short history of a short RNA". Cell 116 (Suppl. 2): S89-92. PMID 15055592.
  • Ruvkun G, Wightman B, Ha I. (2004). "The 20 years it took to recognize the importance of tiny RNAs". Cell 116 (Suppl. 2): S93-6. PMID 15055593.
  • Description of the discovery of lin-4, the first miRNA to be discovered: Lee RC, Feinbaum RL, Ambros V (1993). "The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14". Cell 75 (5): 843-854. PMID 8252621.
  • Definition of miRNA and proposed guidelines to follow in classifying RNA genes as miRNA: Ambros V, Bartel B, Bartel DP, Burge CB, Carrington JC, Chen X, Dreyfuss G, Eddy SR, Griffiths-Jones S, Marshall M, Matzke M, Ruvkun G, Tuschl T (2003). "A uniform system for microRNA annotation". RNA 9 (3): 277-279. PMID 12592000.
  • Jones-Rhoades, Matthew W, Bartel, David P, Bartel, Bonnie (2006). "MicroRNAs AND THEIR REGULATORY ROLES IN PLANTS". Annual Review of Plant Biology 57: 19 - 53.


  • A discussion of the processes that miRNA and siRNAs are involved in, in the context of 2 articles in the same issue of the journal Science: Baulcombe D (2002). "DNA events. An RNA microcosm". Science 297 (5589): 2002-2003. PMID 12242426.


  • An excerpt: "Compare the gene number of fruitfly (13,000) to human (20,000), and it's pretty clear that complexity emerges not just from gene number but from how those genes are regulated. In recent years, it's become increasingly clear that one class of molecules, called microRNAs (miRNAs), exert significant regulatory control over gene expression in most plant and animal species. A mere 22 nucleotides long, miRNAs control a cell's protein composition by preventing the translation of protein-coding messenger RNAs (mRNAs). When a miRNA pairs with an mRNA, through complementary base pairing between the molecules, the mRNA is either destroyed or is not translated....In a new study, Stephen Cohen and his colleagues at the European Molecular Biological Laboratory in Germany establish basic ground rules for miRNA–mRNA pairing using a combination of genetics and computational analyses, and identify different classes of miRNA targets with distinct functional properties....Based on their experimental results, Cohen and colleagues searched the Drosophila genome for biologically relevant targets, and estimate that the fly has about 100 sites for every miRNA in its genome. Since the fruitfly has anywhere from 96 to 124 miRNAs, that means it has 8,000 to 12,000 target sites (in the 11,000 genes sampled). This indicates that miRNAs regulate a large fraction of protein-coding genes. Of the known animal miRNAs, many regulate critical developmental processes. This new approach to predicting targets should help reveal just how much regulatory control actually flows from these tiny bits of RNA."

See also

External links