This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gottwein, E.
Right arrow Articles by Cullen, B. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gottwein, E.
Right arrow Articles by Cullen, B. R.

 Previous Article  |  Next Article 

Journal of Virology, June 2006, p. 5321-5326, Vol. 80, No. 11
0022-538X/06/$08.00+0     doi:10.1128/JVI.02734-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Novel Assay for Viral MicroRNA Function Identifies a Single Nucleotide Polymorphism That Affects Drosha Processing

Eva Gottwein, Xuezhong Cai, and Bryan R. Cullen*

Center for Virology and Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710

Received 29 December 2005/ Accepted 9 March 2006

MicroRNAs (miRNAs) are a class of ~22-nucleotide noncoding RNAs that inhibit the expression of specific target genes at the posttranscriptional level. Recently, 11 miRNAs encoded by the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) were cloned from latently infected cells. While the expression of these miRNAs has been confirmed by Northern analysis, their ability to inhibit target gene expression has not been demonstrated. We have devised a novel assay for miRNA function that uses lentiviral indicator vectors carrying two perfectly complementary target sites for each given miRNA in the 3' untranslated region of the Renilla luciferase gene. This assay allowed us to demonstrate the activity of each viral miRNA upon cotransduction of cells with the Renilla luciferase indicator vector together with a firefly luciferase control vector. In KSHV-infected BC-1 and BCBL-1 cells, but not uninfected control cells, Renilla luciferase expression was selectively reduced up to 10-fold. Interestingly, one of the viral miRNAs (miR-K5) exhibited much higher activity in BC-1 cells than in BCBL-1 cells. Sequence analysis of both viral genomes revealed a single nucleotide polymorphism in the miR-K5 precursor stem-loop, which inhibits the expression of mature miR-K5 in BCBL-1 cells. We show that the primary miR-K5 sequence present in BCBL-1 results in diminished processing by Drosha both in vivo and in vitro. This is the first report of a naturally occurring sequence polymorphism in an miRNA precursor that results in reduced processing and therefore lower levels of mature miRNA expression and function.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, Box 3025, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-3369. Fax: (919) 681-8979. E-mail: culle002{at}mc.duke.edu.

Journal of Virology, June 2006, p. 5321-5326, Vol. 80, No. 11
0022-538X/06/$08.00+0     doi:10.1128/JVI.02734-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Sun, G., Yan, J., Noltner, K., Feng, J., Li, H., Sarkis, D. A., Sommer, S. S., Rossi, J. J. (2009). SNPs in human miRNA genes affect biogenesis and function. RNA 15: 1640-1651 [Abstract] [Full Text]  
  • Horikawa, Y., Wood, C. G., Yang, H., Zhao, H., Ye, Y., Gu, J., Lin, J., Habuchi, T., Wu, X. (2008). Single Nucleotide Polymorphisms of microRNA Machinery Genes Modify the Risk of Renal Cell Carcinoma. Clin. Cancer Res. 14: 7956-7962 [Abstract] [Full Text]  
  • Ye, Y., Wang, K. K., Gu, J., Yang, H., Lin, J., Ajani, J. A., Wu, X. (2008). Genetic Variations in MicroRNA-Related Genes Are Novel Susceptibility Loci for Esophageal Cancer Risk. Cancer Prevention Research 1: 460-469 [Abstract] [Full Text]  
  • Edwards, R. H., Marquitz, A. R., Raab-Traub, N. (2008). Epstein-Barr Virus BART MicroRNAs Are Produced from a Large Intron prior to Splicing. J. Virol. 82: 9094-9106 [Abstract] [Full Text]  
  • Jazdzewski, K., Murray, E. L., Franssila, K., Jarzab, B., Schoenberg, D. R., de la Chapelle, A. (2008). Common SNP in pre-miR-146a decreases mature miR expression and predisposes to papillary thyroid carcinoma. Proc. Natl. Acad. Sci. USA 105: 7269-7274 [Abstract] [Full Text]  
  • Lu, J., Fu, Y., Kumar, S., Shen, Y., Zeng, K., Xu, A., Carthew, R., Wu, C.-I (2008). Adaptive Evolution of Newly Emerged Micro-RNA Genes in Drosophila. Mol Biol Evol 25: 929-938 [Abstract] [Full Text]  
  • Yang, H., Dinney, C. P., Ye, Y., Zhu, Y., Grossman, H. B., Wu, X. (2008). Evaluation of Genetic Variants in MicroRNA-Related Genes and Risk of Bladder Cancer. Cancer Res. 68: 2530-2537 [Abstract] [Full Text]  
  • Ehrenreich, I. M., Purugganan, M. D. (2008). Sequence Variation of MicroRNAs and Their Binding Sites in Arabidopsis. Plant Physiol. 146: 1974-1982 [Abstract] [Full Text]  
  • O'Hara, A. J., Vahrson, W., Dittmer, D. P. (2008). Gene alteration and precursor and mature microRNA transcription changes contribute to the miRNA signature of primary effusion lymphoma. Blood 111: 2347-2353 [Abstract] [Full Text]  
  • Gandy, S. Z., Linnstaedt, S. D., Muralidhar, S., Cashman, K. A., Rosenthal, L. J., Casey, J. L. (2007). RNA Editing of the Human Herpesvirus 8 Kaposin Transcript Eliminates Its Transforming Activity and Is Induced during Lytic Replication. J. Virol. 81: 13544-13551 [Abstract] [Full Text]  
  • Cai, X., Cullen, B. R. (2007). The imprinted H19 noncoding RNA is a primary microRNA precursor. RNA 13: 313-316 [Abstract] [Full Text]  
  • GOTTWEIN, E., CAI, X., CULLEN, B.R. (2006). Expression and Function of MicroRNAs Encoded by Kaposi's Sarcoma-associated Herpesvirus. Cold Spring Harb Symp Quant Biol 71: 357-364 [Abstract]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»