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. 1999 Sep;5(9):1200–1209. doi: 10.1017/s1355838299990246

mRNA localization signals can enhance the intracellular effectiveness of hammerhead ribozymes.

N S Lee 1, E Bertrand 1, J Rossi 1
PMCID: PMC1369843  PMID: 10496221

Abstract

Subcellular localization signals for several mRNAs are positioned in their 3' untranslated regions (UTR). We have utilized the human alpha- and beta-actin 3' UTRs as signals for colocalizing hammerhead ribozymes with a lacZtarget mRNA. Ribozyme and target genes containing matched or unmatched 3' UTRs were cotransfected into 12-day-old chicken embryonic myoblast and fibroblast (CEMF) cultures and assayed by in situ hybridization (ISH) using a dual label, antibody sandwich procedure, and dual fluorescence microscopy to monitor intracellular colocalization. Beta-galactosidase localization in transfectants was visualized by incubation with X-gal and also quantitated by an o-nitrophenyl beta-D-galactopyranoside (ONPG) assay. We found that the percentage of colocalization using the matched alpha- or beta-actin 3' UTR (alpha-alpha or beta-beta) was enhanced approximately threefold relative to unmatched 3' UTRs. The increase in ribozyme-mediated inhibition of beta-galactosidase activity observed when matched 3' UTRs were used was consistent with the observed percentage of colocalization. These results represent the first direct demonstration that mRNA localization signals (zipcodes) can be utilized to enhance intracellular ribozyme efficacy.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ainger K., Avossa D., Morgan F., Hill S. J., Barry C., Barbarese E., Carson J. H. Transport and localization of exogenous myelin basic protein mRNA microinjected into oligodendrocytes. J Cell Biol. 1993 Oct;123(2):431–441. doi: 10.1083/jcb.123.2.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bashirullah A., Cooperstock R. L., Lipshitz H. D. RNA localization in development. Annu Rev Biochem. 1998;67:335–394. doi: 10.1146/annurev.biochem.67.1.335. [DOI] [PubMed] [Google Scholar]
  3. Berleth T., Burri M., Thoma G., Bopp D., Richstein S., Frigerio G., Noll M., Nüsslein-Volhard C. The role of localization of bicoid RNA in organizing the anterior pattern of the Drosophila embryo. EMBO J. 1988 Jun;7(6):1749–1756. doi: 10.1002/j.1460-2075.1988.tb03004.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bertrand E., Pictet R., Grange T. Can hammerhead ribozymes be efficient tools to inactivate gene function? Nucleic Acids Res. 1994 Feb 11;22(3):293–300. doi: 10.1093/nar/22.3.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burgin K. E., Waxham M. N., Rickling S., Westgate S. A., Mobley W. C., Kelly P. T. In situ hybridization histochemistry of Ca2+/calmodulin-dependent protein kinase in developing rat brain. J Neurosci. 1990 Jun;10(6):1788–1798. doi: 10.1523/JNEUROSCI.10-06-01788.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Christoffersen R. E., Marr J. J. Ribozymes as human therapeutic agents. J Med Chem. 1995 Jun 9;38(12):2023–2037. doi: 10.1021/jm00012a001. [DOI] [PubMed] [Google Scholar]
  7. Decker C. J., Parker R. Diversity of cytoplasmic functions for the 3' untranslated region of eukaryotic transcripts. Curr Opin Cell Biol. 1995 Jun;7(3):386–392. doi: 10.1016/0955-0674(95)80094-8. [DOI] [PubMed] [Google Scholar]
  8. Driever W., Nüsslein-Volhard C. The bicoid protein determines position in the Drosophila embryo in a concentration-dependent manner. Cell. 1988 Jul 1;54(1):95–104. doi: 10.1016/0092-8674(88)90183-3. [DOI] [PubMed] [Google Scholar]
  9. Ephrussi A., Dickinson L. K., Lehmann R. Oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell. 1991 Jul 12;66(1):37–50. doi: 10.1016/0092-8674(91)90137-n. [DOI] [PubMed] [Google Scholar]
  10. Frigerio G., Burri M., Bopp D., Baumgartner S., Noll M. Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network. Cell. 1986 Dec 5;47(5):735–746. doi: 10.1016/0092-8674(86)90516-7. [DOI] [PubMed] [Google Scholar]
  11. Garner C. C., Tucker R. P., Matus A. Selective localization of messenger RNA for cytoskeletal protein MAP2 in dendrites. Nature. 1988 Dec 15;336(6200):674–677. doi: 10.1038/336674a0. [DOI] [PubMed] [Google Scholar]
  12. Haseloff J., Gerlach W. L. Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature. 1988 Aug 18;334(6183):585–591. doi: 10.1038/334585a0. [DOI] [PubMed] [Google Scholar]
  13. Hill M. A., Gunning P. Beta and gamma actin mRNAs are differentially located within myoblasts. J Cell Biol. 1993 Aug;122(4):825–832. doi: 10.1083/jcb.122.4.825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Incorvaia R., Padgett R. A. Base pairing with U6atac snRNA is required for 5' splice site activation of U12-dependent introns in vivo. RNA. 1998 Jun;4(6):709–718. doi: 10.1017/s1355838298980207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jackson R. J. Cytoplasmic regulation of mRNA function: the importance of the 3' untranslated region. Cell. 1993 Jul 16;74(1):9–14. doi: 10.1016/0092-8674(93)90290-7. [DOI] [PubMed] [Google Scholar]
  16. Jeffery W. R., Tomlinson C. R., Brodeur R. D. Localization of actin messenger RNA during early ascidian development. Dev Biol. 1983 Oct;99(2):408–417. doi: 10.1016/0012-1606(83)90290-7. [DOI] [PubMed] [Google Scholar]
  17. Kim-Ha J., Smith J. L., Macdonald P. M. oskar mRNA is localized to the posterior pole of the Drosophila oocyte. Cell. 1991 Jul 12;66(1):23–35. doi: 10.1016/0092-8674(91)90136-m. [DOI] [PubMed] [Google Scholar]
  18. Kislauskis E. H., Li Z., Singer R. H., Taneja K. L. Isoform-specific 3'-untranslated sequences sort alpha-cardiac and beta-cytoplasmic actin messenger RNAs to different cytoplasmic compartments. J Cell Biol. 1993 Oct;123(1):165–172. doi: 10.1083/jcb.123.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kislauskis E. H., Zhu X., Singer R. H. Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype. J Cell Biol. 1994 Oct;127(2):441–451. doi: 10.1083/jcb.127.2.441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Long R. M., Singer R. H., Meng X., Gonzalez I., Nasmyth K., Jansen R. P. Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA. Science. 1997 Jul 18;277(5324):383–387. doi: 10.1126/science.277.5324.383. [DOI] [PubMed] [Google Scholar]
  21. Macdonald P. M., Struhl G. cis-acting sequences responsible for anterior localization of bicoid mRNA in Drosophila embryos. Nature. 1988 Dec 8;336(6199):595–598. doi: 10.1038/336595a0. [DOI] [PubMed] [Google Scholar]
  22. Melton D. A. Translocation of a localized maternal mRNA to the vegetal pole of Xenopus oocytes. Nature. 1987 Jul 2;328(6125):80–82. doi: 10.1038/328080a0. [DOI] [PubMed] [Google Scholar]
  23. Michienzi A., Conti L., Varano B., Prislei S., Gessani S., Bozzoni I. Inhibition of human immunodeficiency virus type 1 replication by nuclear chimeric anti-HIV ribozymes in a human T lymphoblastoid cell line. Hum Gene Ther. 1998 Mar 20;9(5):621–628. doi: 10.1089/hum.1998.9.5-621. [DOI] [PubMed] [Google Scholar]
  24. Nüsslein-Volhard C., Frohnhöfer H. G., Lehmann R. Determination of anteroposterior polarity in Drosophila. Science. 1987 Dec 18;238(4834):1675–1681. doi: 10.1126/science.3686007. [DOI] [PubMed] [Google Scholar]
  25. Pal B. K., Scherer L., Zelby L., Bertrand E., Rossi J. J. Monitoring retroviral RNA dimerization in vivo via hammerhead ribozyme cleavage. J Virol. 1998 Oct;72(10):8349–8353. doi: 10.1128/jvi.72.10.8349-8353.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rossi J. J., Kierzek R., Huang T., Walker P. A., Itakura K. An alternate method for synthesis of double-stranded DNA segments. J Biol Chem. 1982 Aug 25;257(16):9226–9229. [PubMed] [Google Scholar]
  27. Sarver N., Cantin E. M., Chang P. S., Zaia J. A., Ladne P. A., Stephens D. A., Rossi J. J. Ribozymes as potential anti-HIV-1 therapeutic agents. Science. 1990 Mar 9;247(4947):1222–1225. doi: 10.1126/science.2107573. [DOI] [PubMed] [Google Scholar]
  28. St Johnston D., Driever W., Berleth T., Richstein S., Nüsslein-Volhard C. Multiple steps in the localization of bicoid RNA to the anterior pole of the Drosophila oocyte. Development. 1989;107 (Suppl):13–19. doi: 10.1242/dev.107.Supplement.13. [DOI] [PubMed] [Google Scholar]
  29. St Johnston D. The intracellular localization of messenger RNAs. Cell. 1995 Apr 21;81(2):161–170. doi: 10.1016/0092-8674(95)90324-0. [DOI] [PubMed] [Google Scholar]
  30. Sullenger B. A., Cech T. R. Tethering ribozymes to a retroviral packaging signal for destruction of viral RNA. Science. 1993 Dec 3;262(5139):1566–1569. doi: 10.1126/science.8248806. [DOI] [PubMed] [Google Scholar]
  31. Sun J. S., Manley J. L. A novel U2-U6 snRNA structure is necessary for mammalian mRNA splicing. Genes Dev. 1995 Apr 1;9(7):843–854. doi: 10.1101/gad.9.7.843. [DOI] [PubMed] [Google Scholar]
  32. Sundell C. L., Singer R. H. Requirement of microfilaments in sorting of actin messenger RNA. Science. 1991 Sep 13;253(5025):1275–1277. doi: 10.1126/science.1891715. [DOI] [PubMed] [Google Scholar]
  33. Symons R. H. Small catalytic RNAs. Annu Rev Biochem. 1992;61:641–671. doi: 10.1146/annurev.bi.61.070192.003233. [DOI] [PubMed] [Google Scholar]
  34. Takizawa P. A., Sil A., Swedlow J. R., Herskowitz I., Vale R. D. Actin-dependent localization of an RNA encoding a cell-fate determinant in yeast. Nature. 1997 Sep 4;389(6646):90–93. doi: 10.1038/38015. [DOI] [PubMed] [Google Scholar]
  35. Tiedge H., Fremeau R. T., Jr, Weinstock P. H., Arancio O., Brosius J. Dendritic location of neural BC1 RNA. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2093–2097. doi: 10.1073/pnas.88.6.2093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Uhlenbeck O. C. A small catalytic oligoribonucleotide. Nature. 1987 Aug 13;328(6131):596–600. doi: 10.1038/328596a0. [DOI] [PubMed] [Google Scholar]
  37. Weeks D. L., Melton D. A. A maternal mRNA localized to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGF-beta. Cell. 1987 Dec 4;51(5):861–867. doi: 10.1016/0092-8674(87)90109-7. [DOI] [PubMed] [Google Scholar]
  38. Wilhelm J. E., Vale R. D. RNA on the move: the mRNA localization pathway. J Cell Biol. 1993 Oct;123(2):269–274. doi: 10.1083/jcb.123.2.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wu J. A., Manley J. L. Base pairing between U2 and U6 snRNAs is necessary for splicing of a mammalian pre-mRNA. Nature. 1991 Aug 29;352(6338):818–821. doi: 10.1038/352818a0. [DOI] [PubMed] [Google Scholar]
  40. Zolotukhin S., Potter M., Hauswirth W. W., Guy J., Muzyczka N. A "humanized" green fluorescent protein cDNA adapted for high-level expression in mammalian cells. J Virol. 1996 Jul;70(7):4646–4654. doi: 10.1128/jvi.70.7.4646-4654.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

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