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MicroRNAs: Review of Discovery, Biogenesis, and Research Areas
Allison O'Brien, PhD
MicroRNAs (miRNAs) are small endogenous RNA molecules (~21-25 nt) that regulate gene expression by
targeting one of more mRNAs for translational repression or cleavage. Although the first miRNA was
identified over ten years ago, it is only recently that we have begun to understand the scope and
diversity of these regulatory molecules.
Discovery
In 1993, Lee, Feinbaum, and Ambros discovered that lin-4 in C. elegans did not code for a protein but
instead produced a pair of short RNA transcripts that each regulate the timing of larval development by
translational repression of lin-14, which encodes for a nuclear protein.[1] They postulated the regulation
was due in part to sequence complementarity between lin-4 and unique repeats within the 3' UTR of the
lin-14 mRNA. The downregulation of lin-14 at the end of the first larval stage initiates the
developmental progression into the second larval stage.[1, 2] It was 7 years later that the second
miRNA, let-7, was discovered.[3] The let-7 miRNA, similar to lin-4, also regulated developmental
timing in C. elegans.
Since the discovery of let-7, thousands of miRNAs have been identified in organisms as diverse as viruses,
worms, and primates through random cloning and sequencing or computational prediction.[4-8] The
identified miRNAs are currently curated and annotated at miRBase, hosted by the Sanger Institute as a
publicly available repository. (http://microrna.sanger.ac.uk)
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| Figure 1 |
| siRNA and miRNA pathways in mammalian cells. Numerical arrows designate miRNA pathway, while alphabetical arrows specify siRNA pathway. 1) miRNA gene is transcribed into a primary miRNA transcript (pri-miRNA) 2) Pri-miRNA is cleaved by Drosha to a hairpin pre-miRNAs 3) Pre-miRNA is transported out of the nucleus by exportin-5 4) Pre-miRNA is cleaved by Dicer to form a short double-stranded miRNA duplex 5) miRNA duplex separates into single-stranded mature miRNAs and complexes with a RISC-like structure 6) mRNA binds with miRNA/RISC complex 7) mRNA is translationally repressed. A) dsRNA enters cell B) dsRNA is cleaved by Dicer to form a short double-stranded siRNA duplex C) siRNA duplex separates into single-stranded siRNAs and complexes with a RISC-like structure D) mRNA binds with siRNA/RISC complex E) mRNA is degraded. |
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Biogenesis
miRNAs are double stranded RNAs of 21-25 nt that are derived from endogenously expressed transcripts with characteristic hairpin structures. Figure 1 diagrams the current understanding of the biogenesis of both miRNA and siRNA. The miRNA pathway begins with the transcription of a primary miRNA (pri-miRNA) from a miRNA gene (step 1). The 70-100 nt hairpin RNAs (pri-miRNA) are processed in the nucleus by the ribonuclease Drosha to become precursor miRNA (pre-miRNA) (step 2). Once the pre-miRNAs are transported into the cytoplasm by exportin 5 (step 3), a second ribonuclease, Dicer, digests the pre-miRNAs resulting in a 21-25 nt miRNA (step 4). At this stage, the miRNA binds the RNA-Induced Silencing Complex (RISC), and aligns with the mRNA (step 5-6). Depending on the level of complementarity between the miRNA and the target sequence, the mRNA can either be translationally repressed (partial) or cleaved (identical) (step 7).[9-12] In plants, cleavage appears to be the primary mode of action, while in mammals translational repressions seems to be the key method.[4, 12].
The siRNA pathway is an evolutionarily conserved response triggered by an externally introduced double stranded RNA (dsRNA) (step A).[9] The dsRNA is cleaved by the ribonuclease, Dicer, into small interfering RNAs (siRNA) that are approximately 21-23nt (step B).[9] The siRNA is loaded into an RNA-Induced Silencing Complex (RISC) which facilitates the separation of the two strands and alignment of the siRNA with its appropriate target mRNA (steps C-D). The siRNA has near perfect complementarity with its target mRNA, and the mRNA cleavage is directed at the site of complementarity (step E). Scientific researchers have utilized the siRNA pathway by artificially introducing either dsRNA or siRNA designed to degrade targeted mRNAs. These synthetic silencing reagents are utilized as molecular biology tools for novel gene identification, gene functional analysis, and biological pathways screens.
Research Areas
miRNA Profiling
The identification and characterization of miRNAs is a rapidly growing area of research as miRNAs regulate a variety of processes such as development, cell proliferation and death, and have been recently linked to oncogenesis.[13-15] A typical approach to identifying miRNAs is to monitor the miRNA gene expression profiles. If a miRNA is found to be differentially expressed in a certain tissue or cell type, it may hypothesized to play a regulatory role in specifying tissue or cell identity. Similarly, if a miRNA is expressed at a specific developmental stage, then it may regulate developmental timing.[16] miRNA expression can be profiled by the cloning and sequencing of miRNAs from specific tissues or cells, or at a specific developmental stage, or by microarray analysis.[17, 18] Cloning and sequencing miRNAs is currently the most widely used method, albeit quite laborious, for the discovery of new miRNAs, while microarray analysis is a high throughput method that typically only reveals the expression of known miRNAs.
miRNA Functional Analysis
The study of miRNA function by the suppression of miRNA expression in cells is a rapidly expanding area of research due to the thousands of microRNA that have been identified in plants, animals, and viruses.[19-23] The natural targets of miRNAs remains to be elucidated thus, synthetic inhibitors of miRNA function represent valuable research tools for exploring miRNA function.[24] The activity of a miRNA can be chemically blocked using morpholino phosphorodiamidate anti-sense oligonucleotides complementary to endogenous miRNAs.[25, 26] Additionally, anti-sense locked-nucleic acid (LNA) oligonucleotides, which have increased binding affinities, have been used to block miRNA in human cells.[27] LNA-modified probes are also being used to shed light on the localization and expression patterns of miRNAs.[28, 29]
References
1. Lee, R., R. Feinbaum, and V. Ambros. Cell, 1993. 75(5): p. 843-54.
2. Wightman, B., I. Ha, and G. Ruvkun. Cell, 1993. 75: p. 855-892.
3. Reinhart, B.J., et al. Nature, 2000. 403: p. 901-906.
4. Rhoades, M.W., et al. Cell, 2002. 110(4): p. 513-20.
5. Enright, A., et al. Genome Biol,. 2003. 5(1): p. R1.
6. Krek, A., et al. Nat. Genet., 2005. 37(5): p. 495-500.
7. Kiriakidou, M., et al. Genes & Dev., 2004. 18(10): p. 1165-1178.
8. Lewis, B., C. Burge, and D. Bartel. Cell, 2005. 120(1): p. 15-20.
9. He, L. and G. Hannon. Nat. Rev. Genet., 2004. 5(7): p. 522-31.
10. Pasquinelli, A., S. Hunter, and J. Bracht. Curr. Opin. Genet. Dev., 2005. 15(2): p. 200-5.
11. Kim, V.N. Nat. Rev. Mol. Cell. Biol., 2005. 6(5): p. 376-385.
12. Bartel, D. Cell, 2004. 116(2): p. 281-97.
13. Zhao, Y., E. Samal, and D. Srivastava. Nature, 2005: p. 1-7.
14. Yekta, S., I.H. Shih, and D.P. Bartel. Science, 2004. 304(5670): p. 594-596.
15. Poy, M., et al. Nature, 2004. 432(7014): p. 226-30.
16. Du, T. and P.D. Zamore. Development, 2005. 132(21): p. 4645-4652.
17. Baskerville, S. and D.P. Bartel. RNA, 2005. 11(3): p. 241-247.
18. Barad, O., et al. Genome Res., 2004. 14(12): p. 2486-2494.
19. Lai, E.C. Nat. Genet., 2002. 30(4): p. 363-4.
20. Lai, E.C., B. Tam, and G.M. Rubin. Genes & Dev., 2005. 19(9): p. 1067-1080.
21. Lim, L., et al. Nature, 2005. 433(7027): p. 769-73.
22. Lim, L.P., et al. Science, 2003. 299(5612): p. 1540.
23. Lim, L.P., et al. Genes & Dev., 2003. 17(8): p. 991-3519.
24. Lee, Y.S., et al. J. Biol. Chem., 2005. 280(17): p. 16635-16641.
25. Hutvagner, G., et al. PLoS Biology, 2004. 2(4): p. e98.
26. Meister, G., et al. RNA, 2004. 10(3): p. 544-550.
27. Lecellier, C.-H., et al. Science, 2005. 308(5721): p. 557-560.
28. Valoczi, A., et al. Nucleic Acids Res. Suppl., 2004. 32(22): p. e175.
29. Wienholds, E., et al. Science, 2005. 309(5732): p. 310-311.
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