Abstract
Investigating the cell biology of gene expression requires methodologies for localizing RNA relative to proteins involved in RNA transcription, processing, and export. Adenovirus is an important model system for the analysis of eukaryotic gene expression and is also being used to investigate the organization of gene expression within the nucleus. Here are described the combined in situ hybridization and immunofluorescence staining techniques that have been used to study the localization of viral RNA relative to nuclear structures that contain splicing factors.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Kuersten, S. and Goodwin, E. B. (2005) Linking nuclear mRNP assembly and cytoplasmic destiny. Biol. Cell 97, 469–478.
Berget, S. M., Moore, C., and Sharp, P. A. (1977) Spliced segments at the 5’ terminus of adenovirus 2 late mRNA. Proc. Natl. Acad. Sci. USA 74, 3171–3175.
Chow, L. T., Gelinas, R. E., Broker. T. R., and Roberts, R. J. (1977) An amazing sequence arrangement at the 5’ ends of adenovirus 2 messenger RNA. Cell 12, 1–8.
Proudfoot, N. J., Furger, A., and Dye, M. J. (2002) Integrating mRNA processing with transcription. Cell 108, 501–512.
Proudfoot, N. (2004) New perspectives on connecting messenger RNA 3’ end formation to transcription. Curr. Opin. Cell Biol. 16, 272–278.
Bridge, E. and Pettersson, U. (1996) Nuclear organization of adenovirus RNA biogenesis. Exp. Cell Res. 229, 233–239.
Pombo, A., Ferreira, J., Bridge, E., and Carmo-Fonseca, M. (1994) Adenovirus replication and transcription sites are spatially separated in the nucleus of infected cells. EMBO J. 13, 5075–5085.
Custodio, N., Carvalho, C., Condado, I., Antoniou, M., Blencowe, B. J., and Carmo-Fonseca, M. (2004) In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei. RNA 10, 622–633.
Aspegren, A. and Bridge, E. (2002) Release of snRNP and RNA from transcrip-tion sites in adenovirus-infected cells. Exp. Cell Res. 276, 273–283.
Aspegren, A., Rabino, C., and Bridge, E. (1998) Organization of splicing factors in adenovirus-infected cells reflects changes in gene expression during the early to late phase transition. Exp. Cell Res. 245, 203–213.
Bridge, E., Riedel, K. U., Johansson, B. M., and Pettersson, U. (1996) Spliced exons of adenovirus late RNAs colocalize with snRNP in a specific nuclear domain.J. Cell Biol. 135, 303–314.
Bridge, E., Mattsson, K., Aspegren, A., and Sengupta, A. (2003) Adenovirus early region 4 promotes the localization of splicing factors and viral RNA in late-phase interchromatin granule clusters. Virology 311, 40–50.
Earnshaw, W. C. and Rattner, J. B. (1991) The use of autoantibodies in the study of nuclear and chromosomal organization. Methods in Cell Biol. 35, 135–175.
Zhang, G., Taneja, K. L., Singer, R. H., and Green, M. R. (1994) Localization of pre-mRNA splicing in mammalian nuclei. Nature 372, 809–812.
Zhang, G., Zapp, M. L., Yan, G., and Green, M. R. (1996) Localization of HIV-1 RNA in mammalian nuclei. J. Cell Biol. 135, 9–18.
Gruenewald-Jahno, S., Keesey, J., Leous, M., van Miltonberg, R., and Schroeder, C., eds. (1996) Nonradioactive In Situ Hybridization Application Manual, 2nd ed. Boehringer Mannheim GmbH Biochemica, Mannheim, Germany, pp. 8–56.
Johnson, C. V., Singer, R. H., and Lawrence, J. B. (1991) Fluorescent detection of nuclear RNA and DNA: Implications for genome organization. Methods Cell Biol. 35, 73–99.
Challberg, M. and Kelly, T. (1989) Animal virus DNA replication. Ann. Rev. Biochem. 58, 671–717.
Puvion-Dutilleul, F. and Pichard, E. (1992) Segregation of viral double-stranded and single-stranded DNA molecules in nuclei of adenovirus infected cells as revealed by electron microscope in situ hybridization. Biol. Cell 76, 139–150.
Puvion-Dutilleul, F. and Puvion, E. (1990) Analysis by in situ hybridization and autoradiography of sites of replication and storage of single-and double-stranded adenovirus type 5 DNA in lytically infected HeLa cells. J. Struct. Biol. 103, 280–289.
Puvion-Dutilleul, F., Pédron, J., and Cajean-Feroldi, C. (1984) Identification of intranuclear structures containing the 72K DNA-binding protein of human adenovirus type 5. Eur. J. Cell Biol. 34, 313–322.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Dirks, R. W., van de Rijke, F. M., Fujishita, S., van der Ploeg, M., and Raap, A. K. (1993) Methodologies for specific intron and exon RNA localization in cultured cells by haptenized and fluorochromized probes. J. Cell Sci. 104, 1187–1197.
Jiménez-Garcia, L. F. and Spector, D. L. (1993) In vivo evidence that transcription and splicing are coordinated by a recruiting mechanism. Cell 73, 47–59.
Xing, Y., Johnson, C. V., Dobner, P. R., and Lawrence, J. B. (1993) Higher level organization of individual gene transcription and RNA splicing. Science 259, 1326–1329.
Jiménez-Garcia, L. F., Green, S. R., Mathews, M. B., and Spector, D. L. (1993) Organization of the double-stranded RNA-activated protein kinase DAI and virus-associated VA RNAI in adenovirus-2-infected HeLa cells. J. Cell Sci. 106, 11–22.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc, Totowa, NJ
About this protocol
Cite this protocol
Bridge, E. (2007). Simultaneous Detection of Adenovirus RNA and Cellular Proteins by Fluorescent Labeling In Situ . In: Wold, W.S.M., Tollefson, A.E. (eds) Adenovirus Methods and Protocols. Methods in Molecular Medicine™, vol 131. Humana Press. https://doi.org/10.1007/978-1-59745-277-9_5
Download citation
DOI: https://doi.org/10.1007/978-1-59745-277-9_5
Publisher Name: Humana Press
Print ISBN: 978-1-58829-901-7
Online ISBN: 978-1-59745-277-9
eBook Packages: Springer Protocols