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Technique / Molecular Biology / miRNA microRNA
| microRNAs (miRNA) are single-stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression. |
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Analysis of small endogenous RNAs. (Curr Protoc Mol Biol. 2008 Jan;Chapter 26:Unit26.7.) Lau NC. Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA. Eukaryotic cells express small noncoding RNAs to silence target genes or regulate chromatin domains. MicroRNAs (miRNA) are endogenous small RNAs that are thought to each regulate multiple mRNA targets. To gain a deeper understanding of processes regulated by small noncoding RNAs, techniques are required to identify and detect them. This unit describes standard laboratory methods for and comments about high-throughput technologies for the identification and detection of small RNAs. This unit also outlines a cell-based reporter gene assay for assessing the regulatory potential of a microRNA on a candidate mRNA target. (miRNA or microRNA technique reviews) 1: Nilsen TW. Mechanisms of microRNA-mediated gene regulation in animal cells. Trends Genet. 2007 Mar 15; [Epub ahead of print] 2: Wang Y, Stricker HM, Gou D, Liu L. MicroRNA: past and present. Front Biosci. 2007 Jan 1;12:2316-29. 3: Kosik KS. The neuronal microRNA system. Nat Rev Neurosci. 2006 Dec;7(12):911-20. Review. 4: Engels BM, Hutvagner G. Principles and effects of microRNA-mediated post-transcriptional gene regulation. Oncogene. 2006 Oct 9;25(46):6163-9. Review. 5: Ying SY, Chang DC, Miller JD, Lin SL. MicroRNA protocols. Perspectives. Methods Mol Biol. 2006;342:351-8. Review. 6: Ishizuka A, Saito K, Siomi MC, Siomi H. In vitro precursor microRNA processing assays using Drosophila Schneider-2 cell lysates. Methods Mol Biol. 2006;342:277-86. Review. 7: Chen CH, Guo M, Hay BA. Identifying microRNA regulators of cell death in Drosophila. Methods Mol Biol. 2006;342:229-40. Review. 8: Griffiths-Jones S. miRBase: the microRNA sequence database. Methods Mol Biol. 2006;342:129-38. Review. 9: Smalheiser NR, Torvik VI. Complications in mammalian microRNA target prediction. Methods Mol Biol. 2006;342:115-27. Review. 10: John B, Sander C, Marks DS. Prediction of human microRNA targets. Methods Mol Biol. 2006;342:101-13. Review. 11: Zeng Y, Cullen BR. Recognition and cleavage of primary microRNA transcripts. Methods Mol Biol. 2006;342:49-56. Review. 12: Gregory RI, Chendrimada TP, Shiekhattar R. MicroRNA biogenesis: isolation and characterization of the microprocessor complex. Methods Mol Biol. 2006;342:33-47. Review. 13: Krol J, Krzyzosiak WJ. Structure analysis of microRNA precursors. Methods Mol Biol. 2006;342:19-32. Review. 14: Ying SY, Chang DC, Miller JD, Lin SL. The microRNA: overview of the RNA gene that modulates gene functions. Methods Mol Biol. 2006;342:1-18. Review. 15: Chang K, Elledge SJ, Hannon GJ. Lessons from Nature: microRNA-based shRNA libraries. Nat Methods. 2006 Sep;3(9):707-14. Review. 16: Zhang B, Pan X, Anderson TA. MicroRNA: a new player in stem cells. J Cell Physiol. 2006 Nov;209(2):266-9. Review. 17: le Sage C, Agami R. Immense promises for tiny molecules: uncovering miRNA functions. Cell Cycle. 2006 Jul;5(13):1415-21. Epub 2006 Jul 1. Review. 18: Rajewsky N. microRNA target predictions in animals. Nat Genet. 2006 Jun;38 Suppl:S8-13. Review. 19: Berezikov E, Cuppen E, Plasterk RH. Approaches to microRNA discovery. Nat Genet. 2006 Jun;38 Suppl:S2-7. Review. 20: Kim VN, Nam JW. Genomics of microRNA. Trends Genet. 2006 Mar;22(3):165-73. Epub 2006 Jan 30. Review. 21: Kong Y, Han JH. MicroRNA: biological and computational perspective. Genomics Proteomics Bioinformatics. 2005 May;3(2):62-72. Review. 22: Chen PY, Meister G. microRNA-guided posttranscriptional gene regulation. Biol Chem. 2005 Dec;386(12):1205-18. Review. 23: Pillai RS. MicroRNA function: multiple mechanisms for a tiny RNA? RNA. 2005 Dec;11(12):1753-61. Review. 24: Du T, Zamore PD. microPrimer: the biogenesis and function of microRNA. Development. 2005 Nov;132(21):4645-52. Review. 25: Vaucheret H. MicroRNA-dependent trans-acting siRNA production. Sci STKE. 2005 Sep 6;2005(300):pe43. Review. 26: Wienholds E, Plasterk RH. MicroRNA function in animal development. FEBS Lett. 2005 Oct 31;579(26):5911-22. Epub 2005 Aug 10. Review. 27: Tang G. siRNA and miRNA: an insight into RISCs. Trends Biochem Sci. 2005 Feb;30(2):106-14. Review. 28: Lai EC. Predicting and validating microRNA targets. Genome Biol. 2004;5(9):115. Epub 2004 Aug 31. Review. 29: Nelson P, Kiriakidou M, Sharma A, Maniataki E, Mourelatos Z. The microRNA world: small is mighty. Trends Biochem Sci. 2003 Oct;28(10):534-40. Review. 30: Brennecke J, Cohen SM. Towards a complete description of the microRNA complement of animal genomes. Genome Biol. 2003;4(9):228. Epub 2003 Aug 21. Review. A small fortune: small RNAs involved in the regulation of developmental timing (Coffee Break) Created: June 5, 2002 
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Last update: 
09-May-2008 07:57 am
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Identification of pyruvate kinase type M2 as potential oncoprotein in squamous cell carcinoma of tongue through microRNA profiling. Int J Cancer. 2008 May 7;
Authors: Wong TS, Liu XB, Chung-Wai Ho A, Po-Wing Yuen A, Wai-Man Ng R, Ignace Wei W
MicroRNAs (miRNAs) are noncoding RNAs with specific regulatory role in gene expression. Recent reports suggested their involvement in human malignancies. Currently, there is no information concerning miRNA expression and functions in squamous cell carcinoma (SCC) of tongue. In this study, we evaluated the expression patterns of 156 mature miRNAs in tongue SCC using Taqman-based microRNA assays. Of these 156 miRNAs, miR-133a and miR-133b were significantly reduced in tongue SCC cells in comparison with the paired normal epithelial cells. Tongue SCC cell lines transfected with miR-133a and miR-133b precursors displayed reduction in proliferation rate. In addition, the number of apoptotic cells was increased in response to the introduction of precursors. Computational target gene prediction suggested that both miR-133a and miR-133b are targeting transcript of pyruvate kinase type M2 (PKM2), a potential oncogene in solid cancers. In tongue SCC cell lines, PKM2 expression was reduced in response to miR-133a and miR-133b precursors transfection. Immunohistochemical staining results of tongue SCC tissues suggested that PKM2 was overexpressed in tongue SCC and was associated with the downregulation of miR-133a and miR-133b. Our results suggested that aberrant reduction of miR-133a and miR-133b was associated with the dysregulation of PKM2 in SCC of tongue. (c) 2008 Wiley-Liss, Inc.
Molecular architecture of a miRNA-regulated 3' UTR. RNA. 2008 May 7;
Authors: Didiano D, Hobert O
Animal genomes contain hundreds of microRNAs (miRNAs), small regulatory RNAs that control gene expression by binding to complementary sites in target mRNAs. Some rules that govern miRNA/target interaction have been elucidated but their general applicability awaits further experimentation on a case-by-case basis. We use here an assay system in transgenic nematodes to analyze the interaction of the Caenorhabditis elegans lsy-6 miRNA with 3' UTR sequences. In contrast to many previously described assay systems used to analyze miRNA/target interactions, our assay system operates within the cellular context in which lsy-6 normally functions, a single neuron in the nervous system of C. elegans. Through extensive mutational analysis, we define features in the known and experimentally validated target of lsy-6, the 3' UTR of the cog-1 homeobox gene, that are required for a functional miRNA/target interaction. We describe that both in the context of the cog-1 3' UTR and in the context of heterologous 3' UTRs, one or more seed matches are not a reliable predictor for a functional miRNA/target interaction. We rather find that two nonsequence specific contextual features beyond miRNA target sites are critical determinants of miRNA-mediated 3' UTR regulation. The contextual features reside 3' of lsy-6 binding sites in the 3' UTR and act in a combinatorial manner; mutation of each results in limited defects in 3' UTR regulation, but a combinatorial deletion results in complete loss of 3' UTR regulation. Together with two lsy-6 sites, these two contextual features are capable of imparting regulation on a heterologous 3' UTR. Moreover, the contextual features need to be present in a specific configuration relative to miRNA binding sites and could either represent protein binding sites or provide an appropriate structural context. We conclude that a given target site resides in a 3' UTR context that evolved beyond target site complementarity to support regulation by a specific miRNA. The large number of 3' UTRs that we analyzed in this study will also be useful to computational biologists in designing the next generation of miRNA/target prediction algorithms.
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