Springer Protocols: Abstract: Using Synthetic Precursor and Inhibitor miRNAs to Understand miRNA Function

Using Synthetic Precursor and Inhibitor miRNAs to Understand miRNA Function

By: Lance P. Ford³, Angie Cheng⁴

Abstract

Full Text

Download PDF (511K)

Although the majority of gene function studies center themselves around protein-encoding RNAs, the study of non-protein-encoding RNAs is becoming more widespread because of the discovery of hundreds of small RNA termed micro (mi) RNA that have regulator functions within cells. Currently, over 470 human miRNA genes are predicted to exist and are annotated within the “miRBase” public miRNA database (http://microrna.sanger.ac.uk/). There is no denying that short interfering (si) and short hairpin (sh) RNAs have revolutionized how scientists approach understanding gene function; however, si and shRNAs are not effective for analyzing the function of miRNAs given that miRNAs are typically short (17–24 bases). In turn, new sets of agents that allow for the expression of miRNA above endogenous levels and inhibition of miRNAs have become a valuable technology for the study of these small regulatory RNAs. In this chapter, we provide step-by-step methods on how to utilize synthetic precursor and antisense inhibitor molecules for understanding miRNA function.

Affiliation(s): (3) Bio Scientific Corporation, Austin, TX, USA
(4) Ambion, Applied Biosystems Inc., Austin, TX, USA

Book Title: Post-Transcriptional Gene Regulation

Series: Methods in Molecular Biology | Volume: 419 | Pub. Date: Dec-21-2007 | Page Range: 289-301 | DOI: 10.1007/978-1-59745-033-1_20

Subject: Genetics/Genomics

Key Words: miRNA function - miRNA inhibition - miRNA expression

References

Download References

1.	Lee, R.C., R.L. Feinbaum, and V. Ambros. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993; 75(5): p. 843–54.

2.	Wightman, B., I. Ha, and G. Ruvkun. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 1993; 75(5): p. 855–62.

3.	Ha, I., B. Wightman, and G. Ruvkun. A bulged lin-4/lin-14 RNA duplex is sufficient for Caenorhabditis elegans lin-14 temporal gradient formation. Genes Dev 1996; 10(23): p. 3041–50.

4.	Lewis, B.P., C.B. Burge, and D.P. Bartel. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005; 120(1): p. 15–20.

5.	Lim, L.P., et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 2005; 433(7027): p. 769–73.

6.	Yekta, S., I.H. Shih, and D.P. Bartel. MicroRNA-directed cleavage of HOXB8 mRNA. Science 2004; 304(5670): p. 594–6.

7.	Schramke, V., et al. RNA-interference-directed chromatin modification coupled to RNA polymerase II transcription. Nature 2005; 435(7046): p. 1275–9.

8.	Bagga, S., et al. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 2005; 122(4): p. 553–63.

9.	Jing, Q., et al. Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell 2005; 120(5): p. 623–34.

10.	Johnson, S.M., et al. RAS is regulated by the let-7 microRNA family. Cell 2005; 120(5): p. 635–47.

11.	Bhattacharyya, S.N., et al. Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell 2006; 125(6): p. 1111–24.

12.	Vatolin, S., K. Navaratne, and R.J. Weil. A novel method to detect functional microRNA targets. J Mol Biol 2006; 358(4): p. 983–96.

13.	Vo, N., et al. A cAMP-response element binding protein-induced microRNA regulates neuronal morphogenesis. Proc Natl Acad Sci USA 2005; 102(45): p. 16426–31.

14.	Naguibneva, I., et al. The microRNA miR-181 targets the homeobox protein Hox-A11 during mammalian myoblast differentiation. Nat Cell Biol 2006; 8(3): p. 278–84.

15.	Fazi, F., et al. A minicircuitry comprised of microRNA-223 and transcription factors NFI-A and C/EBPalpha regulates human granulopoiesis. Cell 2005; 123(5): p. 819–31.

16.	Felli, N., et al. MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation. Proc Natl Acad Sci USA 2005; 102(50): p. 18081–6.

17.	Voorhoeve, P.M., et al. A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 2006; 124(6): p. 1169–81.

18.	Krutzfeldt, J., et al. Silencing of microRNAs in vivo with ‘antagomirs’. Nature 2005.

19.	Chen, C.Z., et al. MicroRNAs modulate hematopoietic lineage differentiation. Science 2004; 303(5654): p. 83–6.

20.	Guimaraes-Sternberg, C., et al. MicroRNA modulation of megakaryoblast fate involves cholinergic signaling. Leuk Res 2006; 30(5): p. 583–95.

21.	Guimaraes-Sternberg, C., et al. MicroRNA modulation of megakaryoblast fate involves cholinergic signaling. Leuk Res 2005.

22.	Zeng, Y., X. Cai, and B.R. Cullen. Use of RNA polymerase II to transcribe artificial microRNAs. Methods Enzymol 2005; 392: p. 371–80.

23.	Zeng, Y. and B.R. Cullen. Sequence requirements for micro RNA processing and function in human cells. RNA 2003; 9(1): p. 112–23.

24.	Zeng, Y. and B.R. Cullen. Efficient processing of primary microRNA hairpins by Drosha requires flanking nonstructured RNA sequences. J Biol Chem 2005; 280(30): p. 27595–603.

25.	Zeng, Y., E.J. Wagner, and B.R. Cullen. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell 2002; 9(6): p. 1327–33.

26.	Chen, C.Z. and H.F. Lodish. MicroRNAs as regulators of mammalian hematopoiesis. Semin Immunol 2005; 17(2): p. 155–65.

27.	McManus, M.T., et al. Gene silencing using micro-RNA designed hairpins. RNA 2002; 8(6): p. 842–50.

28.	Boutla, A., C. Delidakis, and M. Tabler. Developmental defects by antisense-mediated inactivation of micro-RNAs 2 and 13 in Drosophila and the identification of putative target genes. Nucleic Acids Res 2003; 31(17): p. 4973–80.

29.	Meister, G., et al. Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. RNA 2004; 10(3): p. 544–50.

30.	Hutvagner, G., et al. Sequence-specific inhibition of small RNA function. PLoS Biol 2004; 2(4): p. E98.

31.	Cheng, A.M., et al. Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis. Nucleic Acids Res 2005; 33(4): p. 1290–7.

32.	Ford, L.P. Using synthetic miRNA mimics for diverting cell fate: a possibility of miRNA-based therapeutics? Leuk Res 2006; 30(5): p. 511–3.

32a.	Garzon, R., et al. MicroRNA fingerprints during human megakaryocytopoiesis. Proc Natl Acad Sci U S A 2006; 103(13): p. 5078–83.

32b.	Hornstein, E., et al. The microRNA miR-196 acts upstream of Hoxb8 and Shh in limb development. Nature 2005; 438(7068): p. 671–4.

Comments (Loading...)

Post comment/View all comments