MicroRNA: Small regulators with global impact
MicroRNAs (miRNAs) are an important class of small RNA molecules that are expressed in eukaryotes. Although the first miRNA was identified in a genetic screen in 1993 1, it was not until 2001 that the breadth of the miRNA gene class was recognised with the cloning and sequencing of more than 100 miRNAs from worms, humans and mice 1–4. These evolutionarily conserved, non-coding RNA molecules regulate translation of mRNAs through base pair interactions 5. With some exceptions, worm and human miRNAs inhibit the translation of specific mRNAs, while plant miRNAs induce mRNA degradation.
 In cells, the ~22 nt mature, functional miRNAs are produced by a recently described process (see Figure 1). A primary transcript (pri-mRNA) that can be more than 1000nt in length is first produced in the nucleus. An RNA hairpin (precursor miRNA, pre-miR) comprising ~80nt, including the mature miRNA, results from digestion of pri-miRNA by the double-strand-specific ribonuclease, Drosha 6. The resulting pre-miRNA is transported to the cytoplasm via a process that involves Exportin-5 7,8. The pre-miRNA is further digested by Dicer 6,8 to generate a short, partially double-stranded RNA wherein one strand is the mature miRNA. The mature miRNA is taken up by a protein complex that is similar to, if not identical to, the RNA-induced silencing complex (RISC) that supports RNA interference (RNAi) 9,10 and the bound complex functions to regulate translation.
As a unique class of small RNA molecules, miRNAs require special tools for accurate and sensitive analysis. Ambion’s scientists have developed a portfolio of products that provide a complete solution to rapidly identifying and characterising miRNA. The most recent addition is the Pre-miR™ miRNA precursor molecules and Anti-miR™ miRNA inhibitors for the regulation of miRNA expression in cells.
MicroRNA functional analysis can be performed with protocols that are similar to standard genes. Up-regulation of the miRNAs can be conducted to identify gain-of-function phenotypes; down-regulation or inhibition can be conducted to identify loss-of-function phenotypes. The combination of up- and down-regulation can be used to identify genes that are regulated by specific miRNAs, as well as to identify cellular processes that are affected by specific miRNAs. Key applications include:
- miRNA target site validation
- miRNA target site identification
- Screening for miRNAs that regulate the expression of a gene
- Screening for miRNAs that affect a cellular process
 The pMIR-REPORT™ miRNA expression reporter vector provides accurate, quantitative, in-cell measurement of miRNA expression. This miRNA-validated reporter system contains Luciferase, under the control of a mammalian promoter/terminator system, with a miRNA-cloning region following the termination of translation sequence. This vector is designed for the cloning of specific, putative sequence binding sites for miRNA. The pMIR-REPORT miRNA expression reporter vector can also be used as a screening tool, in which random or non-random sequences are inserted into the reporter to identify miRNA targets, or coupled with a library of miRNA that can be used to screen a target sequence with all known miRNAs.
The use of these new tools can help lead advances in therapeutics as well as basic research into the functioning of development and protein expression. Look to Ambion as the complete miRNA solution provider.
Footnotes
1.Lee R, Feinbaum R, and Ambros V (1993) The heterochronic gene lin-4 of C. elegans encodes small RNAs with antisense complementarity to lin-14. Cell 75: 843-54
2. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T (2001) Identification of novel genes coding for small expressed RNAs. Science 294: 853-8
3. Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294: 858-62
4. Lee RC and Ambros V (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294: 862-4
5. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: 281-97
6. Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, Lee J, Provost P, Radmark O, Kim S, Kim VN (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425: 415-9
7. Lund E, Guttinger S, Calado A, Dahlberg JE, Kutay U (2004) Nuclear export of microRNA precursors. Science 303(5654): 95-8
8. Yi R, Qin Y, Macara IG, Cullen BR (2004) Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17(24): 3011-6
9. Hutvagner G, Zamore PD (2002) A microRNA in a multiple-turnover RNAi enzyme complex. Science 297(5589): 2056-60
10. Mourelatos Z, Dostie J, Paushkin S, Sharma A, Charroux B, Abel L, Rappsilber J, Mann M, Dreyfuss G (2002) miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev 16(6): 720-8
 < Back to Drug Discovery index
< Back to Market Resources index |
